Abstract:

Peptide arrays and uses thereof for diagnostics, therapeutics and
research. Ultra high density peptide arrays are generated using
photolithography, such as using photoresist techniques.

Claims:

1. A peptide array comprising: a plurality of peptides coupled to a
support; wherein at least a set of said peptides comprise sequences
identical to a predetermined sequence with the exception of one monomer;
wherein said one monomer is in a different position within each of said
peptides.

2. A peptide array comprising: a plurality of peptides coupled to a
support; wherein at least a set of peptides have a first monomer in
position X; and wherein said set comprises one or more of the following
elements:(a) at least 1000 of said different peptides;(b) each of said
different peptides is located within a feature with an area of up to 1
um2; or(c) each of said different peptides has at least 20 monomers.

3. A peptide array comprising: a plurality of peptides coupled to a
support; wherein at least a set of said peptides has a sequence derived
from a common protein sequence with at least one phosphoacceptor; wherein
each of said peptides has a sequence that overlaps with the sequence of
at least one other peptide in said set; wherein said array comprises one
or more of the following elements:(a) at least 1000 of said different
peptides;(b) each of said different peptides is located within a feature
with an area of up to 1 um2; or(c) each of said different peptides
has at least 20 monomers.

4. A peptide array comprising: a plurality of peptides coupled to a
support; wherein a set of said peptides comprises at least one
phosphoacceptor; wherein said array comprises one or more of the
following elements:(a) at least 4000 different peptides;(b) each
different peptide is located within a feature with an area of up to 1
um2;(c) each peptide has at least 20 monomers;(d) the array is
produced by photolithography using photomasks.

5. The peptide array of claim 3 or 4, wherein the phosphoacceptor is a
Ser, Thr, Tyr, or derivative thereof.

6. The peptide array of claim 3 or 4, wherein the phosphoacceptor is
phosphorylated or unphosphorylated.

7. The peptide array of claim 1 or 2, wherein said one monomer is an amino
acid.

8. The peptide array of claim 1 or 2, wherein the one monomer is a
phosphoacceptor.

9. The peptide array of claim 1 or 2, wherein the one monomer is
phosphorylated or unphosphorylated.

10. The peptide array of claim 1 or 2, wherein the one monomer is a Ser,
Thr, Tyr, or derivative thereof.

11. The peptide array of any one of claims 1-4, wherein said peptides
comprise phosphoacceptors for at least 50% of all the kinases of a kinase
family.

12. The peptide array of any one of claims 1-4, wherein said peptides
comprise phosphoacceptors for at least 50% of all the kinases of an organ
or organism.

13. The peptide array of claim 12, wherein said organ is a liver, kidney
or heart.

14. The peptide array of claim 12, wherein said organism is a eukaryote or
prokaryote.

15. The peptide array of claim 12, wherein said organism is a human.

16. The peptide array of any one of claims 1-4, wherein said peptides
comprise phosphoacceptors for at least 50% of all the phosphatases of an
organ or organism.

17. The peptide array of claim 16, wherein said organ is a liver, kidney
or heart.

18. The peptide array of claim 16, wherein said organism is a eukaryote or
prokaryote.

19. The peptide array of claim 16, wherein said organism is a human.

20. The peptide array of any one of claims 1-4, wherein said peptides are
comprised of at least 5 monomers.

21. The peptide array of any one of claims 1-4, wherein said set of
peptides is comprised of at least 2 different peptides.

22. The peptide array of any one of claims 1-4, wherein said array
contains at least 5 sets of peptides.

23. The peptide array of any one of claims 1-4, wherein up to 70% of said
peptides are full-length compared to predetermined sequences used to
design said peptides.

24. The peptide array of any one of claims 1-4, wherein up to 80% of said
peptides are identical to predetermined sequences used to design said
peptides.

25. The peptide array of any one of claims 1-4, wherein said array has at
least 5000, 10,000, 100,000, 1,000,000, 2,000,000, 3,000,000, 10,000,000,
20,000,000, or 100,000,000 different peptides.

26. The peptide array of any one of claims 1-4, wherein each peptide is
located within a feature that has an area of up to 1 um2.

[0002]Screening mechanisms to identify peptides binding domains (e.g.,
enzyme substrates, therapeutic peptides, etc.) are extremely valuable.
While there are some peptide arrays available commercially, such spotted
arrays have low density and relatively low fidelity. Thus, there is a
need for a better high density, high fidelity, robust system for
analyzing peptides and the proteome.

SUMMARY OF THE INVENTION

[0003]The present invention relates to compositions and methods for
creating peptide arrays using photolithography and methods of using the
peptide arrays produced by photolithography. The peptide arrays of the
present invention can be produced by photoresist technology. In general,
the invention features peptide arrays containing kinase or phosphatase
substrates.

[0004]The inventions described herein include those disclosed in U.S.
Provisional Application Ser. Nos. 60/941,413 filed on Jun. 1, 2007 and
61/035,727 filed on Mar. 11, 2008, both of which hereby are incorporated
in their entirety by reference.

[0005]Implementation of the invention can include one or more of the
following features.

[0006]In general, in one aspect, a peptide array is provided including a
plurality of peptides coupled to a support, wherein at least a set of the
peptides can include sequences identical to a predetermined sequence with
the exception of one monomer, wherein the one monomer is in a different
position within each of the peptides.

[0007]In general, in another aspect, a peptide array is provided including
a plurality of peptides coupled to a support, wherein at least a set of
peptides can have a first monomer in position X, and wherein the set can
include one or more of the following elements: at least 1000 different
peptides; each of the different peptides can be located within a feature
with an area of up to 1 um2; or each of the different peptides can have
at least 20 monomers. X can be any amino acid in a sequence.

[0008]In general, in yet another aspect, a peptide array is provided
including a plurality of peptides coupled to a support; wherein at least
a set of the peptides can have a sequence derived from a common protein
sequence with at least one phosphoacceptor; wherein each of said peptides
can have a sequence that overlaps with the sequence of at least one other
peptide in said set; wherein the array can include one or more of the
following elements: at least 1000 of the different peptides; each of the
different peptides can be located within a feature with an area of up to
1 um2; or each of said different peptides can have at least 20 monomers.

[0009]In general, in yet another aspect, a peptide array is provided
including a plurality of peptides coupled to a support, wherein a set of
said peptides can include at least one phosphoacceptor; wherein said
array comprises one or more of the following elements at least 4000
different peptides, each different peptide can be located within a
feature with an area of up to 1 um2, each peptide can have at least 20
monomers, and the array can be produced by photolithography using
photomasks.

[0010]The phosphoacceptor can be a Ser, Thr, Tyr, or derivative thereof.
The phosphoacceptor can be phosphorylated or unphosphorylated. The said
one monomer can be an amino acid. The one monomer can be a
phosphoacceptor. The one monomer can be phosphorylated or
unphosphorylated.

[0011]The one monomer can be a Ser, Thr, Tyr, or derivative thereof.

[0012]The peptides can include phosphoacceptors for at least 50% of all
the kinases of a kinase family.

[0013]The peptides can include phosphoacceptors for at least 50% of all
the kinases of an organ or organism. The organ can be a liver, kidney or
heart. The organism can be a eukaryote or prokaryote. The organism can be
a human.

[0014]The peptides can include phosphoacceptors for at least 50% of all
the phosphatases of an organ or organism. The organ can be a liver,
kidney or heart. The organism can be a eukaryote or prokaryote. The
organism can be a human. The peptides can include at least 5 monomers.

[0015]The set of peptides can include at least 2 different peptides. The
array can contain at least 5 sets of peptides. Up to 70% of said peptides
can be full-length compared to predetermined sequences used to design
said peptides. Up to 80% of the peptides can be identical to
predetermined sequences used to design said peptides.

[0016]The array can have at least 5000, 10,000, 100,000, 1,000,000,
2,000,000, 3,000,000, 10,000,000, 20,000,000, or 100,000,000 different
peptides. Each peptide can be located within a feature that has an area
of up to 1 um2.

INCORPORATION BY REFERENCE

[0017]All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same extent as
if each individual publication or patent application was specifically and
individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]The novel features of the invention are set forth with particularity
in the appended claims. A better understanding of the features and
advantages of the present invention will be obtained by reference to the
following detailed description that sets forth illustrative embodiments,
in which the principles of the invention are utilized, and the
accompanying drawings of which:

[0019]FIG. 1 illustrates steps for in situ synthesis of peptides on a
support using photolithography and photoresist.

[0028]FIG. 10 illustrates peptides that form a substrate peptide cluster.
Each peptide represents the sequence of a peptide in a feature that forms
the peptide cluster. Each sequence has a single Ser, Thr, or Tyr, as
represented by the dark circles. The Ser, Thr, or Tyr is in a different
monomer position for each peptide in the cluster. The other surrounding
amino acids remain the same between all peptides within the cluster.

[0029]FIG. 11 illustrates one peptide sequence that is part of a substrate
peptide cluster. Each peptide sequence has a single Ser, Thr, or Try in
position 5.

[0030]FIG. 12 illustrates peptides that form a substrate peptide cluster,
wherein each peptide represents the monomer sequence of a feature that
forms the peptide cluster. The peptide sequences are derived from a known
sequence and overlap with other peptide sequences in the peptide cluster
that also represent a portion of the known, or common sequence.

[0031]FIG. 13A) is a schematic of a sample with a mixture of kinases used
in a kinase assay with the peptide array; B) is a graph showing that Src
kinase and Abl kinase in the same sample do not interfere with each other
and can be used in the same kinase assay.

[0032]FIG. 14 illustrates a peptide sequence consisting of 9 monomers for
a kinase peptide array and a signal for detection of phosphorylation.

[0035]FIG. 17A) shows the peptide arrays that detect Abl, Src, or both;
and a chart showing the signal to noise ratio (SNR). B) is a graph
depicting detection of WT kinase activity compared to and mutant kinase
and background using peptide arrays.

[0036]FIG. 18 depicts graphs along with the peptide arrays from which the
data was obtained. PKA and PKB, kinases of the same family, have
different activity against specific peptide substrates. The kinases show
a difference in preferred specificity in position -4 (4 amino acids
shifted from the phosphorylation site, Serine "S"), -4 (one position from
phosphorylation site), and +1 (one position from the serine).

[0037]FIG. 19: depicts graphs along with the peptide arrays from which the
data was obtained. PKC has a different sequence preference in comparison
to PKA and PKB. PKC shows a different preference in position -4, (4 amino
acids shifted from the phosphorylation site, Serine "S") and +1 (one
position from the serine).

[0038]FIG. 20 depicts the positional preference of the AGC family kinases
PKA, PKB, and PKC. The preference was based on relative signal intensity
over kemptide. The bolded residues are from previously published work
whereas the other residues were not published.

[0039]FIG. 21 is a graph showing a peptide array kinase inhibition assay.
The ATP competitive inhibitor, staurosporin ("Stau.") inhibited Src
kinase activity by up to 80%. The IC50 was estimated to be approximately
450 nM.

[0040]FIG. 22 depicts Gleevac inhibition on different forms of Abl kinase.
Gleevac inhibition of phosphorylated Abl kinase, non phosphorylated Abl
kinase, and Src kinase, or both, was tested using peptide arrays with Abl
and Src substrates. A) Gleevac does not have an effect on phosphorylated
Abl kinase nor Src kinase activity. B) Gleevac inhibits the activity of
non phosphorylated Abl kinase. C) The peptide arrays used for testing
kinase activity of phosphorylated Abl and Src, with or without Gleevac.
D) The peptide arrays used for testing kinase activity of non
phosphorylated Abl and Src, with or without Gleevac. E) A chart showing
the percent inhibition of Gleevac.

[0041]FIG. 23 shows the specificity of different kinase inhibitors on Abl
and Src. Activity is measured using peptide arrays with Abl and Src
peptide substrates.

[0042]FIG. 24 depicts a schematic of a peptide on an array with a cleavage
site and fluorophore for use in cleavage assays.

[0043]FIG. 25 shows a graph of the cleavage assay for trypsin. The
sequence of the substrate is depicted below the graph.

[0044]FIG. 26 shows the fluorescence intensity of the peptide array before
and after assay with HIV-1 protease. The peptide substrate is shown above
the graphs, the cleavage site is in bold.

[0046]The present invention relates to peptide arrays, methods of
manufacturing peptides arrays, and various applications of such peptide
arrays. Peptide arrays are preferably generated using one or more of the
methods described below.

Methods of Manufacturing Peptide Arrays

Overview of Photolithography and In Situ Peptide Synthesis on a Support

[0047]The peptides of the arrays of the present invention are synthesized
in situ on a support. In some instances, the peptide arrays are made
using photolithography. Photolithography involves the use of
microfabrication to selectively remove parts of a thin film (or the bulk
of a support). Light can be used to transfer a geometric pattern from a
photomask (or mask) to a light-sensitive chemical (e.g., photoresist) on
the support. A series of chemical treatments then engraves the exposure
pattern into the material underneath the photoresist, examples of which
are described herein.

[0048]To achieve spatially defined combinatorial polymer synthesis on a
support surface, masks can be used to control radiation or light exposure
to specific locations on a surface provided with linker molecules
containing radiation (or photo)-labile protecting groups. In the exposed
locations, the radiation-labile protecting groups are removed. The
surface is then contacted with a solution containing a monomer. The
monomer can have at least one site that is reactive with the newly
exposed reactive moiety on the linker and at least a second reactive site
protected by one or more radiation-labile protecting groups. The desired
monomer is then coupled to the unprotected linker molecules. The process
can be repeated to synthesize a large number of polymers in specific
locations on a support (See, for example, U.S. Pat. No. 5,143,854 to
Pirrung et al., U.S. Patent Application Publication Nos. 2007/0154946
(filed on Dec. 29, 2005), 2007/0122841 (filed on Nov. 30, 2005),
2007/0122842 (filed on Mar. 30, 2006), and 2008/0108149 (filed on Oct.
23, 2006).

Maskless Photolithography Using Micromirrors

[0049]An alternative to photolithographic masks is the use of
micromirrors, which comprises an array of switchable optical elements
such as a two-dimensional array of electronically addressable. Projection
optics focuses an image of the micromirrors on the support where the
reactions for polymers are conducted. Under the control of a computer,
each of the micromirrors is selectively switched between a first position
at which it projects light on the substrate through the optical system
and a second position at which it deflects light away from the substrate.
The plurality of small and individually controllable rocking-mirrors can
steer light beams to produce images or light patterns. Reactions at
different regions on the solid support can be modulated by providing
irradiation of different strengths using such micromirror device, or
digital micromirror device (DMD), which is a programmable photoreaction
optical device.

[0050]Micromirror devices are available commercially, such as Texas
Instruments' digital light projector (DLP). The controlled light
irradiation allows control of the reactions to proceed at a desirable
rate. Such devices are discussed for example, in Hornbeck, L. J.,
"Digital light processing and MEMS, reflecting the digital display needs
of the networked society," SPIE Europe Proceedings, 2783, 135-145 (1996),
U.S. Pat. Nos. 5,096,279, 5,535,047, 5,583,688 and 5,600,383. Other types
of electronically controlled display devices may be used for generating
light patterns. For example, a reflective liquid crystal array display
(LCD) device, commercially available from a number of companies, such
Displaytech, Inc. Longmont, Colo. USA, can contain a plurality of small
reflectors with a liquid crystal shutter placed in front of each
reflector to produce images or light patterns. A transmissive LCD display
can also be used to generate light patterns. A transmissive LCD display
containing a plurality of liquid crystal light valves have valves that
are on, so light passes; and when a liquid crystal light valve is off,
light is blocked. Therefore, a transmissive LCD display can be used in
the same way as an ordinary photomask is used in a standard
photolithography process (L. F. Thompson et al., "Introduction to
Microlithography", American Chemical Society, Washington, D.C. (1994)).
See also Gao et al. "Light directed massively parallel on-chip synthesis
of peptide arrays with t-Boc chemistry" Proteomics 2003, 3, 2135-2141 and
Ishikawa (WO/2000/003307) "MASKLESS PHOTOLITHOGRAPHY SYSTEM".

In Situ Peptide Synthesis on a Solid Support

[0051]In some instances, photoresist and photolithography are used for the
in situ synthesis of peptides on a support, as illustrated in FIG. 1.
First, linker molecules with protecting groups are attached to a solid
support. Next, photoresist is applied to the surface of the support
(100). The photoresist layer can include a polymer, a photosensitizer,
and a photo-active agent. Photoresist can be applied by a spin-coating
method, and the photoresist-coated support can then be baked. Baking
promotes removal of excess solvent from the photoresist and provides for
a uniform film. Next, a photomask is placed over the photoresist layer to
restrict regions that will be exposed to radiation (120). Radiation is
then transmitted through the photomask onto the photoresist layer (120).
Radiation exposure of the photoresist results in reagents that can cleave
the protecting groups from molecules. The cleaving reagent may be
generated owing to absorption of light by a photosensitizer followed by
reaction of the photosensitizer with the cleavage reagent precursor,
energy transfer from the photosensitizer to the cleavage reagent
precursor, or a combination of two or more different mechanisms.

[0052]Protecting groups are cleaved from the molecules in areas that were
exposed to radiation, whereas the protecting groups will not be cleaved
from molecules that were not exposed. Removal of protecting groups can be
accelerated by heating (baking) the support after the radiation exposure.

[0053]After radiation exposure, the photoresist is removed (140).
Deprotected molecules are available for further reaction whereas
molecules that retain their protective groups are not available for
further reaction (160). The processes may be repeated to form polymers on
the support surface (180) (see also, e.g., U.S. Pat. No. 5,677,195 to
Winkler et al.).

Supports

[0054]The solid support, or support, refers to a material or group of
materials having a rigid or semi-rigid surface or surfaces. In some
aspects, at least one surface of the solid support will be substantially
flat, although in some aspects it may be desirable to physically separate
synthesis regions for different molecules with, for example, wells,
raised regions, pins, etched trenches, or the like. In certain
embodiments, the solid support may be porous.

[0055]Support materials useful in embodiments of the present invention
include, for example, silicon, bio-compatible polymers such as, for
example poly(methyl methacrylate) (PMMA) and polydimethylsiloxane (PDMS),
glass, SiO2 (such as, for example, a thermal oxide silicon wafer such as
that used by the semiconductor industry), quartz, silicon nitride,
functionalized glass, gold, platinum, and aluminum. Functionalized
surfaces include for example, amino-functionalized glass, carboxy
functionalized glass, and hydroxy functionalized glass. Additionally, a
support may optionally be coated with one or more layers to provide a
surface for molecular attachment or functionalization, increased or
decreased reactivity, binding detection, or other specialized
application. Support materials and or layer(s) may be porous or
non-porous. For example, a support may be comprised of porous silicon.
Additionally, the support may be a silicon wafer or chip such as those
used in the semiconductor device fabrication industry. In the case of a
wafer or chip, a plurality of arrays may be synthesized on the wafer. A
person skilled in the art would know how to select an appropriate support
material.

Linker Molecules

[0056]The peptides present on the array may be linked covalently or
non-covalently to the array, and can be attached to the array support
(e.g., silicon or other relatively flat material) by cleavable linkers. A
linker molecule can be a molecule inserted between the support and
peptide that is being synthesized, and a linker molecule may not
necessarily convey functionality to the resulting peptide, such as
molecular recognition functionality, but instead elongates the distance
between the support surface and the peptide functionality to enhance the
exposure of the peptide functionality on the surface of the support.
Preferably a linker should be about 4 to about 40 atoms long to provide
exposure. The linker molecules may be, for example, aryl acetylene,
ethylene glycol oligomers containing 2-10 monomer units (PEGs), diamines,
diacids, amino acids, among others, and combinations thereof. Examples of
diamines include ethylene diamine and diamino propane. Alternatively, the
linkers may be the same molecule type as that being synthesized (i.e.,
nascent polymers), such as polypeptides and polymers of amino acid
derivatives such as for example, amino hexanoic acids. A person skilled
in the art would know how to design appropriate linkers.

Monomers

[0057]The monomers used for peptide synthesis can include amino acids. In
some instances all peptides on an array are composed of naturally
occurring amino acids. In others, peptides on an array can be composed of
a combination of naturally occurring amino acids and non-naturally
occurring amino acids. In other cases, peptides on an array can be
composed solely from non-naturally occurring amino acids. Non-naturally
occurring amino acids include peptidomimetics as well as D-amino acids.
The R group can be found on a natural amino acid or a group that is
similar in size to a natural amino acid R group. Additionally, unnatural
amino acids, such as β-alanine, phenylglycine, homoarginine,
aminobutyric acid, aminohexanoic acid, aminoisobutyric acid,
butylglycine, citrulline, cyclohexylalanine, diaminoproprionic acid,
hydroxyproline, norleucine, norvaline, ornithine, penicillamine,
pyroglutamic acid, sarcosine, and thienylalanine can also be incorporated
by the embodiments of the invention. These and other natural and
unnatural amino acids are available from, for example, EMD Biosciences,
Inc., San Diego, Calif.

Protecting Groups

[0058]The unbound portion of the linker molecule, or free end of the
linker molecule, can have a reactive functional group which is blocked,
protected or otherwise made unavailable for reaction by a removable
protective group. The protecting group can be bound to a monomer, a
polymer, a linker molecule or a monomer, or polymer, or a linker molecule
attached to a solid support to protect a reactive functionality on the
monomer, polymer, or linker molecule. Protective groups that may be used
in accordance with an embodiment of the invention include all acid and
base labile protecting groups. For example, peptide amine groups can be
protected by t-butoxycarbonyl (t-BOC or BOC) or benzyloxycarbonyl (CBZ),
both of which are acid labile, or by 9-fluorenylmethoxycarbonyl (FMOC),
which is base labile.

[0060]Photoresist formulations useful in the present invention can include
a polymer, a solvent, and a radiation-activated cleaving reagent. Useful
polymers include, for example, poly(methyl methacrylate) (PMMA),
poly-(methyl isopropenyl ketone) (PMPIK), poly-(butene-1-sulfone) (PBS),
poly-(trifluoroethyl chloroacrylate) (TFECA), copolymer-(α-cyano
ethyl acrylate-α-amido ethyl acrylate (COP), and poly-(2-methyl
pentene-1-sulfone). Useful solvents include, for example, propylene
glycol methyl ether acetate (PGMEA), ethyl lactate, and ethoxyethyl
acetate. The solvent used in fabricating the photoresist may be selected
depending on the particular polymer, photosensitizer, and photo-active
compound that are selected. For example, when the polymer used in the
photoresist is PMMA, the photosensitizer is isopropyl-thioxanthenone, and
the photoactive compound is diphenyliodonium chloride, PGMEA or ethyl
lactate may be used as the solvent.

[0061]In exemplary photoresist formulations, the mass concentration of the
polymer may between about 5% and about 50%, the mass concentration of a
photosensitizer may be up to about 20%, the mass concentration of the
photo-active compound may be between about 1% and 10%, the balance
comprising a suitable solvent. After the photoresist is deposited on the
support, the support typically is heated to form the photoresist layer.
Any method known in the art of semiconductor fabrication may be used to
for depositing the photoresist solution. For example, the spin coating
method may be used in which the support is spun typically at speeds
between about 1,000 and about 5,000 revolutions per minute for about 30
to about 60 seconds. The resulting wet photoresist layer has a thickness
ranging between about 0.1 μm to about 2.5 μm.

[0062]In some instances the photoresist can include radiation-activated
catalysts (RAC), or more specifically photo activated catalysts (PACs).
Photosensitive compounds act as catalysts to chemically alter synthesis
intermediates linked to a support to promote formation of polymer
sequences. Alternatively, RACs can activate an autocatalytic compound
which chemically alters the synthesis intermediate in a manner to allow
the synthesis intermediate to chemically combine with a later added
synthesis intermediate or other compound. For example, one or more linker
molecules are bound to or otherwise provided on the surface of a support.

[0065]Optionally, the photoresists useful in the present invention may
also include a photosensitizer. In general, a photosensitizer absorbs
radiation and interacts with the RAC, such as PAG, through one or more
mechanisms, including, energy transfer from the photosensitizer to the
cleavage reagent precursor, thereby expanding the range of wavelengths of
radiation that can be used to initiate the desired catalyst-generating
reaction. As such, the photosensitizer can be a radiation sensitizer,
which is any material that shifts the wavelengths of radiation required
to initiate a desired reaction. Useful photosensitizers include, for
example, benzophenone and other similar diphenyl ketones, thioxanthenone,
isopropylthioxanthenone, anthraquinone, fluorenone, acetophenone, and
perylene. Thus, the photosensitizer allows the use of radiation energies
other than those at which the absorbance of the radiation-activated
catalyst is non-negligible.

[0066]The present invention may also further include the presence of an
enhancer that is ester labile to acid catalyzed thermolytic cleavage,
itself produces an acid, enhancing the removal of protective groups. The
enhancer can be any material that amplifies a radiation-initiated
chemical signal so as to increase the effective quantum yield of the
radiation. Enhancers include, but are not limited to, catalytic
materials. The use of an enhancer in radiation-assisted chemical
processes is termed chemical amplification. Chemical amplification has
many benefits. Non limiting examples of the benefit of chemical
amplification include the ability to decrease the time and intensity of
irradiation required to cause a desired chemical reaction. Chemical
amplification also improves the spatial resolution and contrast in
patterned arrays formed using this technique.

[0067]The enhancer is a compound or molecule that can be added to a
photoresist in addition to a radiation-activated catalyst. An enhancer
can by activated by the catalyst produced by the radiation-induced
decomposition of the RAC and autocatalyticly reacts to further (above
that generated from the radiation-activated catalyst) generate catalyst
concentration capable of removing protecting groups. For example, in the
case of an acid-generating RAC, the catalytic enhancer can be activated
by acid and or acid and heat and autocatalyticly reacts to form further
catalytic acid, that is, its decomposition increases the catalytic acid
concentration. The acid produced by the catalytic enhancer removes
protecting groups from the growing polymer chain.

[0068]FIG. 2 shows the photogeneration of an acid and the deprotection of
an amine group of a surface-attached amino acid. A support surface is
provided having a first amino acid attached to the surface. In this
example, the first amino acid is N-protected with a t-BOC
(tert-butoxycarbonyl) protecting group. The support surface is coated
with a photoresist, and in this example the photoresist contains the
phoactivated acid generator triaryl sulfonium hexafluoroantimonatate
(TASSbF6). Upon exposure to radiation, an acid is produced in the
photoresist and the N-protecting group is removed from the attached
peptide in the region of UV exposure.

[0069]FIG. 3 illustrates means of photo-acid generation (PAG). Acids can
be generated photochemically. Alternatively, the cleaving reagent may be
generated owing to absorption of light by a photosensitizer followed by
reaction of the photosensitizer with the cleavage reagent precursor,
energy transfer from the photosensitizer to the cleavage reagent
precursor, or a combination of two or more different mechanisms.

Deprotection and Coupling

[0070]Using the techniques disclosed herein, it is possible to
advantageously irradiate relatively small and precisely known locations
on the surface of the support (e.g., within 1 μm2 or 0.5
μm2). The radiation does not directly cause the removal of the
protective groups, such as through a photochemical reaction upon
absorption of the radiation by the synthesis intermediate or linker
molecule itself, but rather the radiation acts as a signal to initiate a
chemical catalytic reaction which removes the protective group in an
amplified manner. Therefore, the radiation intensity as used in the
practice of the present invention to initiate the catalytic removal by a
catalyst system of protecting groups can be much lower than, for example,
direct photo removal, which can result in better resolution when compared
to many non-amplified techniques.

[0071]Acids or bases can be used to remove the protective group, and the
functional group is made available for reaction, i.e. the reactive
functional group is unblocked. A PAC is located or otherwise provided on
the surface of the support in the vicinity of the linker molecules, for
example in a photoresist layer coating the support. The PAC by itself or
in combination with additional catalytic components is referred to herein
as a catalyst system. Using lithographic methods and techniques well
known to those of skill in the art, a set of first selected regions on
the surface of the support can be exposed to radiation of certain
wavelengths. The radiation activates the PAC which then either directly
or through an autocatalytic compound catalytically removes the protecting
group from the linker molecule making it available for reaction with a
subsequently added synthesis intermediate. The autocatalytic compound can
then undergo a reaction producing at least one product that removes the
protective groups from the linker molecules in the first selected
regions.

[0072]In one embodiment, the RAC produces an acid when exposed to
radiation, the monomer can be an amino acid containing an acid removable
protecting group at its amino or carboxy terminus, and the linker
molecule terminates in an amino or carboxy acid group bearing an acid
removable protective group. The embodiment may further include the
presence of an enhancer that is ester labile to acid catalyzed
thermolytic cleavage, itself produces an acid, enhancing the removal of
protective groups.

[0073]The use of PACs and autocatalytic compounds initiates a chemical
reaction which catalyzes the removal of a large number of protective
groups. With the protective groups removed, the reactive functional
groups of the linker molecules are made available for reaction with a
subsequently added synthesis intermediate or other compound. The support
is then washed or otherwise contacted with an additional synthesis
intermediate that reacts with the exposed functional groups on the linker
molecules to form a sequence. In this manner, a sequence of monomers of
desired length can be created by stepwise irradiating the surface of the
support to initiate a catalytic reaction to remove a protective group
from a reactive functional group on a already present synthesis
intermediate and then introducing a monomer, i.e. a synthesis
intermediate, that will react with the reactive functional group, and
that will have a protective group for later removal by a subsequent
irradiation of the support surface.

[0074]Accordingly, a second set of selected regions on the support which
may be the same or different from the first set of selected regions on
the support is, thereafter, exposed to radiation and the removable
protective groups on the synthesis intermediates or linker molecules are
removed. The support is then contacted with an additional subsequently
added synthesis intermediate for reaction with exposed functional groups.
This process is repeated to selectively apply synthesis intermediates
until polymers of a desired length and desired chemical sequence are
obtained. Protective groups on the last added synthesis intermediate in
the polymer sequence can then be optionally removed and the sequence is,
thereafter, optionally capped.

[0075]FIGS. 4A and B illustrate the stepwise in situ synthesis efficiency
for the synthesis of a penta glycine peptide. FIG. 4A shows the step wise
percentage yield for synthesizing a penta glycine peptide using the
photoactive layer formulation with optimized resist at 50 mJ was about
96-98% at each step. FIG. 4B illustrates fluorescence intensity at each
step. In some instances, up to 20%, 30%, 40%, 50%, 60%, 70%, or 80% of
the peptides on an array are the full-length of predetermined sequences.
In some instances, up to 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the
peptides on an array are identical in sequence and length to
predetermined sequences for such peptides.

Generation of Arrays Using Electrochemical Means

[0076]In addition to photo acid generation, arrays can be constructed that
allow for generation of acids through electrochemical means. High
throughput synthesis of dense molecular arrays can be accomplished
through the use of a solid phase catalytic or amplification layer and an
array of electrodes. Electrochemical reactions generate a catalyst for
protective group removal. A solid phase amplification layer that contains
electro-active species is provided.

[0077]A feature of an array could contain an electrode to generate an
electrochemical reagent, a working electrode to synthesize a polymer, and
a confinement electrode to confine the generated electrochemical reagent.
The electrode to generate the electrochemical reagent could be of any
shape, including, for example, circular, flat disk shaped and hemisphere
shaped.

[0078]A support or silicon wafer can consist of an array of electrodes
that can be fabricated using semiconductor processing methods. A polymer
building block having a protecting group is attached to the solid support
through a linker molecule in a coupling reaction. As discussed more fully
herein, in this example, the linker molecule serves to distance the
polymer from the surface of the chip. In the case of peptide synthesis,
the building block molecule is an amino acid that is protected by, for
example, a tert-butoxycarbonyl group. The surface is initially treated
with oxygen plasma to generate an oxidized metal surface and the linker
is coupled to the oxidized surface. Alternately, the surface may be
coated with a thin porous SiO2 layer and the linker attached through
standard silane coupling chemistry. The surface is then coated with a
thin solid-phase layer that is capable of generating an acid (H+,
protons) when exposed to a voltage of about -2 V to about +2 V, i.e., an
amplification layer. The solid phase amplification layer is composed of
matrix polymer (such as, for example, PMMA) dispersed with
electro-sensitizers (molecules commonly used as redox pairs belonging to
the quinine family such as hydroquinone, benzoquinone). Optionally, the
solid phase layer can also contain amplifier molecules (termed
electro-acid amplifiers (EAA)) that can amplify the generation of protons
from protons generated from electro-sensitizers. The solid phase
amplification layer serves to cleave protecting groups; it can be
activated causing the proximate solid phase layer to generate protons.
The support is baked and the amplification layer is removed leaving two
types of building blocks on the surface: the unmodified protected
building block and the deprotected building block. A second building
block is coupled to the deprotected first building block. This method can
be repeated until the desired polymeric molecule(s) are synthesized on
the support surface.

[0079]Similar approaches can be used for cleaving DMT (dimethoxytrityl)
protecting groups for oligo nucleotide synthesis. Also, for base
cleavable protecting groups such as F-moc groups, bases can be generated
electrochemically along with base amplifiers (such as particular types of
carbamates) in the solid phase layer for deprotection chemistry. This
approach can also be used for small molecule synthesis (molecules having
a molecular weight of less than about 800) generally done using
principles currently applied in solution phase electrochemistry.

[0080]The polymer molecules can be built upon a support that contains an
array of individually addressable electrodes. A protected spacer molecule
is coupled to the surface of the support. By selectively activating
regions of the array, the protected molecule attached to the surface is
prepared for coupling a second molecule through the removal of its
protecting group. A protected polymer building block is coupled to the
deprotected surface-attached molecule. By repeatedly activating and
deprotecting regions of the surface of the support building block
molecules are coupled to the surface of the support in a spatially
specific manner.

[0081]Electro-sensitizers (electroactive compounds) are compounds or
molecules that can generate protons (H+) upon exposure to electrons.
A chemical reaction may be used to generate protons in a solid-phase
electroactive layer upon activation by an applied voltage.
Electro-sensitizers that are dispersed in the solid phase amplification
layer can be, for example, molecules commonly used as redox pairs
belonging to the quinine family, such as, hydroquinone and benzoquinone.

[0082]Optionally, the amplification layer may also contain amplifier
compounds that amplify the generation of protons from protons generated
from electro-sensitizers (acid amplifier compounds). These amplifier
molecules can be chosen from a class of molecules such as acid amplifiers
(class of sulfonates undergoing autocatalytic fragmentation), photoacid
generators such as, for example, onium salts such as diaryliodonium and
triarylsulphonium salts, thermal acid generators, such as for example,
2,4,4,6-tetrabromocyclohexadienone, benzoin tosylate, 2-nitrobenzyl
tosylate and other alkyl esters of organic sulfonic acids. The
heat-catalyzed removal of a t-butyl group produces propene and protons.

[0083]The electrodes that may be used in embodiments of the invention may
be composed of, but are not limited to, metals such as iridium and/or
platinum, and other metals, such as, palladium, gold, silver, copper,
mercury, nickel, zinc, titanium, tungsten, aluminum, as well as alloys of
these metals, and other conducting materials, such as, carbon, including
glassy carbon, reticulated vitreous carbon, basal plane graphite, edge
plane graphite, and graphite. Doped oxides such as indium tin oxide, and
semiconductors such as silicon oxide and gallium arsenide are also
contemplated. Additionally, the electrodes may be composed of conducting
polymers, metal doped polymers, conducting ceramics and conducting clays.

[0084]The electrode(s) may be connected to an electric source in any known
manner. For example, connecting the electrodes to the electric source may
include CMOS (complementary metal oxide semiconductor) switching
circuitry, radio and microwave frequency addressable switches, light
addressable switches, direct connection from an electrode to a bond pad
on the perimeter of a semiconductor chip, or combinations thereof. CMOS
switching circuitry involves the connection of each of the electrodes to
a CMOS transistor switch. The switch could be accessed by sending an
electronic address signal down a common bus to SRAM (static random access
memory) circuitry associated with each electrode. When the switch is on,
the electrode is connected to an electric source. Radio and microwave
frequency addressable switches involve the electrodes being switched by a
RF or microwave signal. This allows the switches to be thrown both with
and/or without using switching logic. The switches can be tuned to
receive a particular frequency or modulation frequency and switch without
switching logic. Light addressable switches are switched by light. In
this method, the electrodes can also be switched with and without
switching logic. The light signal can be spatially localized to afford
switching without switching logic. This could be accomplished, for
example, by scanning a laser beam over the electrode array; the electrode
being switched each time the laser illuminates it.

[0085]The generation of and electrochemical reagent of a desired type of
chemical species requires that the electric potential of the electrode
that generates the electrochemical reagent have a certain value, which
may be achieved by specifying either the voltage or the current. The
desired potential at an electrode may be achieved by specifying a desired
voltage value or the current value such that it is sufficient to provide
the desired voltage. The range between the minimum and maximum potential
values is determined by the type of electrochemical reagent chosen to be
generated.

[0086]A wafer is a semiconductor support. A wafer could be fashioned into
various sizes and shapes. It could be used as a support for a microchip.
The support could be overlaid or embedded with circuitry, for example, a
pad, via, an interconnect or a scribe line. The circuitry of the wafer
could also serve several purposes, for example, as microprocessors,
memory storage, and/or communication capabilities. The circuitry can be
controlled by the microprocessor on the wafer itself or controlled by a
device external to the wafer.

[0087]A via interconnection refers to a hole etched in the interlayer of a
dielectric which is then filled with an electrically conductive material,
for example, tungsten, to provide vertical electrical connection between
stacked up interconnect metal lines that are capable of conducting
electricity. A scribe line is typically an inactive area between the
active dies that provide area for separating the die. Often metrology and
alignment features populate this area.

[0088]Array chips on silicon wafers can be built using silicon process
technology and SRAM like architecture with circuitries including
electrode arrays, decoders, and serial-peripheral interface, for example.
Individually addressable electrodes can be created with CMOS circuitry.
The CMOS circuitry, among other functions, amplifies the signal, and
reads and writes information on the individually addressable electrodes.
A CMOS switching scheme can individually address different working
electrodes on a wafer. Each die pad on the die can branch into a large
array of synthesis electrodes. CMOS switches ensure that a given
electrode (or an entire column, or an entire row) can be modified one
base pair at a time.

[0089]Voltage source and counter electrode (plating tool) are shown to
complete the electrical circuit. The electrodes of the array can
electrically connect through a CMOS switch through a bonding pad to a
voltage source. A counter electrode is also supplied. With this scheme,
and electrode can be individually activated. The bonding pad is used, for
example, for power and signal delivery. The die pads can be
interconnected by either using a multilevel interconnect (two or more
layers) across a scribe line on the front side of the wafer or by using a
via interconnect that traverses from the front side of the wafer to the
backside of the wafer.

[0090]The use of photolithography, e.g., with photoresist and RAC, or the
other manufacturing means described herein, allows for arrays that
provide that each polymer or peptide with a distinct sequence can be
synthesized within a feature with an area between 0.2 to 100 um2,
0.2 to 10 um2, 0.2 to 1 um2, 0.2 to 0.5 um2, or in an area
of up to 0.5, 1, 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000 um2.

[0091]The arrays of the present invention have several advantageous
features. The arrays are made using a scalable process using standard
semiconduct fabrication tools. Each process step is precisely controlled
and reproducible, resulting in a robust array. Array synthesis is highly
automated and optimized to significantly reduce process variation. The
peptide arrays of the present invention allow high-throughput use, can be
reliable, and can be cost-efficient.

[0092]Alternative embodiments to the methods described above for
generating peptide array using photoresist-RAC may be found in, for
example, U.S. Pat. Nos. 6,083,697 and 6,770,436 to Beecher et al. and
U.S. Patent Application Publication Nos. 2007/0154946 (filed on Dec. 29,
2005), 2007/0122841 (filed on Nov. 30, 2005), and 2007/0122842 (filed on
Mar. 30, 2006).

Characteristics of the Peptide Arrays

[0093]The peptide arrays of the present invention can include any one or
more of the characteristics described herein, and such arrays can be
manufactured using any of the means described herein.

Peptide Arrays with Enzyme Substrates

[0094]In some instances, a peptide array of the present invention, e.g.,
one constructed using photolithography comprises peptides that are enzyme
substrates. Thus, a subset of the peptides on the array or all of the
peptides on the array may be enzymatic substrates.

[0095]The enzymatic substrates (e.g., peptides) on the array can be
physiological (naturally occurring sequences), artificial, or a
combination thereof. Examples of physiological peptides include peptide
substrates that are naturally occurring or a fragment of a physiological
protein. Examples of artificial peptides can include randomly synthesized
peptides, peptides designed based on physiological substrates, and
peptides designed based on the structure or known binding of enzymes. In
some embodiments, the peptide array can be a mix of artificial and
physiological substrates.

[0096]A peptide array can be designed to provide specific information
about the enzymes for the user. For example, a peptide array can provide
information on all known enzymes, all enzymes of a specific class (e.g.,
kinases, or hydrolases, such as phosphatases, and proteases), all known
enzymes in a specific pathway(s) (e.g., PKC, p53, TRAIL, TNFR1, and JNK),
or all known substrates of a single enzyme.

[0097]Alternatively, information can be provided for a subset of enzymes
in a specific class (for example, a specific kinase family such as casein
kinases or AGC kinases), a subset of enzymes in a pathway, or a subset of
substrates of an enzyme. In some instances, a peptide array comprises a
plurality of peptides that collectively represent all known physiological
kinase substrates for a specific kinase, e.g., ATM. In another
embodiment, a peptide array comprises a plurality of peptides that
collectively represent all physiological substrates for an entire class
of enzymes, e.g., serine phosphatases. For example, the peptide array can
comprise protease or phosphatase substrate peptides for at least 50%,
90%, 99%, or all of the phosphatase substrates, or kinase substrates of
an organ or organism. Furthermore, the peptide array can comprise kinase
substrate peptides for at least 50%, 90%, 99%, or all of the kinase
substrates an organ or an organism, for example, kinase substrates for
the kinome of an organism, such as publicly available at
www.kinase.com/mammalian.

[0098]At least a subset or all peptides on a peptide array of the present
invention can be substrates for enzymes in a biological pathway. For
example, at least a subset of peptides on a peptide array can be
substrates of enzymes in DNA damage signaling pathways. Other biological
pathways whose substrates can be represented on an array can include
apoptosis signaling pathways, G protein-coupled receptor (GPCR) signaling
pathway, or pathways involved in diseases or conditions, such as a
disease associated with apoptosis, a disease associated with signal
transduction pathways of GPCRs, cancer, inflammation, neurodegenerative
diseases, and Alzheimer's disease. For example, the peptides on the array
can be peptides or peptide fragments of molecules involved in
physiological cellular process, such as in signaling pathways involved in
GPCR signaling (for example, as seen in FIGS. 5A-C), or peptides that
represent sequences of proteins that are downstream of a G-protein
coupled receptor. In other embodiments, a peptide array comprises
substrates that are peptides or peptide fragments of molecules involved
in DNA damage signaling (for example, in FIG. 6), apoptosis (for example,
FIG. 7), or peptides or peptide fragments of proteins involved in cancer,
inflammation, or neurodegenerative diseases (for example, in FIG. 8), and
Alzheimer's (for example, in FIG. 9).

[0099]A peptide array can comprise peptides that are substrates for
hydrolases. For example, an array can have at least a subset of its
peptides be substrates of esterases such as nucleases,
phosphodiesterases, lipases, phosphatases, glycosylases, etherases,
proteases, or acid anhydride hydrolases, (e.g. helicases and GTPases).
Other hydrolases whose substrates can be found on a peptide array of the
invention include enzymes that hydrolyze ether bonds, non-peptide
carbon-nitrogen bonds, halide bonds, phosphorus-nitrogen bonds,
sulfur-nitrogen bonds, carbon-phosphorus bonds, sulfur-nitrogen bonds,
carbon-phosphorus bonds, sulfur-sulfur bonds, and carbon-sulfur bonds.
Additional examples of hydrolases include acetylesterase, thioesterase,
and sulfuric ester hydrolases.

[0100]In one embodiment, a set of peptides on an array can include
protease sites for at least 50% of all the proteases of a protease
family. In another embodiment, a set of peptides on an array can comprise
protease sites for at least 50% of all the proteases of an organ or
organism. In another embodiment, a set of peptides on an array can
include protease sites for at least 50% of all the proteases of the
liver, kidney, or heart. A set of peptides on an array can include
protease sites for at least 50% of all the proteases of a eukaryote or
prokaryote. A set of peptides on an array can include protease sites for
at least 50% of all the proteases of a human.

[0101]In one embodiment, the present invention contemplates a peptide
array produced by photolithography using any of the means described
herein, wherein the array comprises a plurality of peptides that are
protease substrates. The proteases that these peptides act as substrates
to include serine proteases, threonine proteases, cysteine proteases,
aspartic acid proteases, metalloproteases, and glutamic acid proteases.
Substrates to proteases such as those described in the peptidase
database, http://merops.sanger.ac.uk/ can be used in the present
invention.

[0102]Examples of phosphatases whose substrates can be generated as
natural or artificial peptides include tyrosine-specific phosphatases,
serine/threonine specific phosphatases, dual specificity phosphatases,
histidine phosphatases, and lipid phosphatases. Additional phosphatases
whose substrates can be inserted into any of the peptide arrays herein
include those described in the kinase-phosphatase database,
http://www.proteinlounge.com/kinase_phosphate.asp. For example,
substrates to alkaline phophastase and/or PP2A can be provided on any of
the peptide arrays described herein.

[0103]The peptide arrays can also comprise substrates for kinases, such as
kinases described in the kinase-phosphatase database,
http://www.proteinlounge.com/kinase_phosphate.asp, or the human kinome,
for example at www.kinase.com/mammalian.

[0104]The peptides on a peptide array can be organized in peptide
clusters. The peptide array can have at least a subset of peptides form
one or more peptide clusters, or all of the peptides form one or more
peptide clusters. Each peptide in a peptide cluster can be the same or
different.

[0105]A peptide array can have at least 1, 2, 5, 10, 20, 50, 75, 100,
1000, or 10,000 peptide clusters. The number of different peptides (or
features) in a cluster can be from 2 to 100,000,000. In some embodiments,
a cluster has at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
different peptides (or features). In other embodiments, the peptide
cluster has hundreds or thousands of different peptides (or features),
for example at least 100, 200, 300, 500, 1000, 1500, 2000, 5000, 10,000,
15,000, or 150,000 different peptides (or features). Each of the features
can have a different peptide sequence, or a subset of the features have
the same peptide sequence.

[0106]In some embodiments, the different peptides (or features) within a
peptide cluster all comprise peptides with one or more enzymatic reaction
sites. For example, all peptide clusters include different peptides with
hydrolase sites, such as a site for a phosphatase or protease, to
dephosphorylate or cleave the peptide, respectively, or phosphorylation
sites to phosphorylate the peptide. In some embodiments, each peptide may
have a single enzymatic reaction site. The enzymatic reaction site can be
the same for all different peptides in the cluster. For example, a
peptide substrate cluster can have 10,000 different peptides each with a
phosphorylation site. The peptide sequence of a peptide may be the same,
or different, monomer sequence as the peptide sequences of other peptides
in the peptide cluster.

[0107]A peptide array can also comprise a peptide cluster wherein each
peptide of the peptide cluster comprises an enzymatic reaction site, such
as a hydrolase or phosphorylation site, at a different position in the
peptide sequence. For example, the enzymatic site of peptides within in a
feature is at a different position than the monomer sequence of peptides
in another feature within the same peptide cluster, wherein the remaining
sequence of the peptides in both features is identical to a single
predetermined sequence (see FIG. 10). A peptide cluster such as described
above, for example, can comprise at least 9 features, wherein each
feature comprises a peptide sequence different than the other. Each row
of monomers as shown in FIG. 10 represents the peptide sequence of a
given feature. The predetermined sequence is identical with the exception
of the amino acid sequence shift of one, from one peptide sequence in to
another peptide sequence. The single enzymatic reaction site is shown as
a single dark. The enzymatic reaction site is in a different position in
each of the 9 monomer sequences. The remaining monomers are the same for
each of the peptides, and this peptide substrate cluster of 9 different
monomer sequences. Variations of this substrate peptide cluster is
obvious to one of ordinary skill in the arts, for example, substrate
clusters with less than 9 monomer sequences, such as a cluster with 5
peptide sequences, the peptides being 5 monomers long, and the peptide
sequence differing from others within the peptide cluster by one amino
acid shift. In other embodiments, the substrates clusters have monomer
sequences at least 9, 10, 11, 12, 13, 14, 15, 18, or 20 monomers long,
with the corresponding number of unique peptide sequences and features in
a peptide cluster. In some embodiments, the features are up to 1 um and
the peptide arrays comprise at least 1000, 2000, 3000, 4000, or 5000
features. Each of the features can have a unique peptide sequence, or a
subset of the features have the same peptide sequence. It is well known
to one of skill in the arts, enzymatic reactions sites can encompass any
sites recognized by an enzyme, and variations of the peptide clusters,
for example, the number of monomers of a peptide, the number of peptide
sequences in the cluster, and the variations of predetermined sequences
can be designed. The peptide clusters can be used to determine the ideal
in vitro substrate for an enzyme, for example, the best in vitro kinase
substrate.

[0108]In other embodiments, the single enzymatic reaction site can be in
the same monomer position as all the other peptide sequences in a peptide
cluster, for example, as seen in FIG. 11, wherein the single enzymatic
reaction site is a phosphorylation site, such as Ser, Thr, or Tyr, in
position 5. The remaining monomer positions for example in positions 1 to
4, and 6 to 9, can be any amino acid. The number of unique peptide
sequences in this embodiment can encompass all the different variations.
In other embodiments, the enzymatic reaction site can be a hydrolase
site, such as a protease or phosphatase site. In other embodiments, each
peptide in a cluster has at least 9, 10, 11, 12, 13, 14, 15, 18, or 20
monomers. In some embodiments, the features are up to 1 um2 and the
peptide arrays comprise at least 1000, 2000, 3000, 4000, or 5000
features. Each of the features can have a unique peptide sequence, or a
subset of the features have the same peptide sequence. It is well known
to one of skill in the arts, enzymatic reactions sites can encompass any
sites recognized by an enzyme, and variations of the peptide clusters,
for example, the number of monomers of a peptide, number of peptides in
the cluster, and the number of variations for random amino acids in the
monomer positions not encompassing the enzymatic reaction site can be
designed. The peptide clusters can be used to determine the ideal in
vitro substrate for an enzyme, for example, the best in vitro kinase
substrate.

[0109]In other embodiments, the peptide sequences in a peptide cluster are
derived from a protein sequence, wherein each peptide sequence overlaps
with another peptide sequence in the substrate cluster, such that each
peptide sequence is a portion or fragment of a common or known protein
sequence (e.g. FIG. 12). The known protein sequence has at least one
reaction site. In some embodiments, the known protein sequence has at
least 2, 3, 4, 5, 6, 7 or 8 reaction sites. The reaction sites can be a
hydrolase site, such as a protease or phosphatase site, or a
phosphorylation site. The known protein sequence can also have a mixture
of enzymatic reactions sites, for example, both protease and
phosphorylation sites. The peptide sequences that are derived from the
known protein sequence can have no reaction sites, at least 1 reaction
site, or at least 2, 3, 4, 5, 6, 7 or 8 reaction sites. The overlap of
monomers between the peptide sequences can be at least 1 monomer, or at
least 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19
monomers. The number of unique peptide sequences in this embodiment can
encompass coverage of the entire common protein sequence, or a portion of
the entire common protein sequence. The substrates clusters can have
monomer sequences at least 9, 10, 11, 12, 13, 14, 15, 18, or 20 monomers
long, with the corresponding number of unique peptide sequences and
features in a peptide cluster. In some embodiments, the features are up
to 1 um2 and the peptide arrays comprise at least 1000, 2000, 3000,
4000, or 5000 features. Each of the features can have a unique peptide
sequence, or a subset of the features have the same peptide sequence. It
is well known to one of skill in the arts, enzymatic reactions sites can
encompass any sites recognized by an enzyme, and variations of the
peptide clusters, for example, the number of monomers of a peptide,
number of peptides in the cluster, and the number of variations for the
peptide sequences will vary depending on the common protein sequence. The
peptide clusters can be used to map the position of the enzymatic site
for a given enzyme.

[0110]Peptide arrays with kinase substrates can be used for drug
development. Samples from targeted tissues/cells can be applied to a
peptide array with kinase substrates, and the phosphorylation of
substrates can reveal a "kinase activity fingerprint". Peptide substrate
phosphorylation and a "kinase activity fingerprint" can be used to yield
information on target validation, hits/leads generation, lead
optimization, preclinical animal studies (pharmacokinetic (PK),
pharmacodynamic (PD) and toxicity), and Phase I/II/III clinical trials.
Peptide substrate phosphorylation can also be used to study side effects
of treatments on organs (e.g. heart, kidney, or liver).

[0111]The present invention also provides peptide arrays and uses of
peptide arrays in research applications and diagnostics.

Peptide Arrays with Peptides from Proteomes

[0112]The arrays of the present invention can contain at least a set of
peptides that cover an entire proteome (set of proteins expressed by a
genome) of a cell, tissue, organ, or organism. The sets of peptides can
cover the proteome on a single chip or on more than one chip. The sets of
peptides that comprise the entire proteome can be on at least 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 chips. The number of chips needed to cover the
entire proteome can be dependent on the number of features on the chips.

[0113]The organism can be a eukaryote or a prokaryote. The organism can be
an animal, plant, or fungus. The organism can be a human or yeast. The
peptide array can contain all the antigenic peptides from a human
proteome. The organism can be an infectious agent, a bacterium, a
microorganism. The sequence of the peptides from a proteome can overlap
and can be antigenic. A set of peptides on the array can have an amino
acid shift of one amino acid position with respect to at least one other
peptide. A set of peptides can have a sequence that overlaps with another
peptide by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, or 19 amino acids. Peptides from proteomes of different
species can be on the same peptide array. The peptides on the array can
be clustered based on whether the organisms belong to separate families.

[0114]Peptides on an array of the present invention can be from animal
organs, including the heart, liver, kidney, brain, skin, lung, stomach,
pancreas, intestines, urinary bladder, uterus, testicles, or spleen.
Peptides on an array of the present invention can be from animal tissues
include, but are not limited to, epithelium, connective tissue, muscle
tissue, and nervous tissue.

[0115]A set of peptides on an array of the present invention can be
derived from vegetative plant organs include root, stem, and leaf. A set
of peptides on an array of the present invention can be from reproductive
plant organs include flower, seed, and fruit. A set of peptides on an
array of the present invention can be from plant tissue includes
epidermis, vascular tissue, and ground tissue.

[0116]The arrays of the present invention can contain at least a set of
peptides that cover an entire proteome of a cell, tissue, organ, or
organism can contain at least 10,000 features, individual features with
an area up to 35 um2, or have peptides with up to 500 monomers.

[0118]The peptides on a peptide array can include at least 10,000, 50,000,
500,000, 1,000,000, 2,000,000, 3,000,000, 10,000,000, 20,000,000 or
100,000,000 different peptides.

[0119]A set of peptides on an array can contain predicted MHC class I or
MHC class II binding peptides of an organ or organism. A peptide sequence
can be a predicted to be an MHC class I or MHC class II binding peptide
by a computer program. A peptide sequence can be predicted to be an MHC
class I or MHC class II binding peptide by an experiment. A peptide
sequence can be predicted to be an MHC class I or MHC class II binding
peptide by visual inspection. A predicted MHC class II binding peptide
can be 10-30 monomers long. A predicted MHC class II binding peptide can
be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30 amino acids long. Methods of predicting MHC
class II peptides are known by those skilled in the art.

Peptide Arrays with Peptides from Oncogenes

[0120]An array of the present invention can contain peptides with
sequences from known oncogenes. Examples of oncogenes include MYC, RAS,
WNT, ERK, SRC, ABL, BCL2, and TRK. Additional oncogenes include v-myc,
N-MYC, L-MYC, v-myb, v-fos, v-jun, v-ski, v-rel, v-ets-1, v-ets-2,
v-erbA1, v-erbA2, BCL2, MDM2, ALL1(MLL), v-sis, int2, KS3, HST, EGFR,
v-fms, v-KIT, v-ros, MET, TRK, NEU, RET, mas, SRC, v-yes, v-fgr, v-fes,
ABL, H-RAS, K-RAS, N-RAS, BRAF, gsp, gip, Dbl, Vav, v-mos, v-raf, pim-1,
v-crk. Oncogenes are disclosed in Croce, "Oncogenes and Cancer", The New
England Journal of Medicine, 358; 502-511 and supplemental information
(2008). The peptides of arrays of the present invention can cover the
full-length sequence of known oncogenes. The peptides from known
oncogenes on the array can also overlap in their sequence as is
illustrated in FIG. 12. A set of peptides on the array can have an amino
acid shift of one amino acid position with respect to at least one other
peptide. A set of peptides can have a sequence that overlaps with another
peptide by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, or 19 amino acids. The peptides on the array can comprise the
entire sequence of 10%, 50%, 90%, or all proteins encoded by oncogenes.
The peptides on a peptide array can include at least 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomers. Peptides on an
array can have 6-150 monomers, 6-500 monomers, 3-35 monomers.

[0121]The peptides on a peptide array can include at least 10,000, 50,000,
500,000, 1,000,000, 2,000,000, 3,000,000, 10,000,000, 20,000,000 or
100,000,000 different peptides.

[0122]The sequence of the peptides from oncogenes can be antigenic. A set
of peptides on an array can contain predicted MHC class I or MHC class II
binding peptides from proteins encoded by oncogenes. A peptide sequence
can be a predicted to be an MHC class I or MHC class II binding peptide
by a computer program. A peptide sequence can be predicted to be an MHC
class I or MHC class II binding peptide by an experiment. A peptide
sequence can be predicted to be an MHC class I or MHC class II binding
peptide by visual inspection of the sequence. A predicted MHC class II
binding peptide can be 10-30 monomers long. A predicted MHC class II
binding peptide can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. Methods
of predicting MHC class II peptides are known by those skilled in the
art.

Peptide Arrays with Peptides for the Study and Diagnosis of Autoimmune
Disorders

[0124]Examples of antigens that elicit autoantibodies in autoimmune
disorders have been described in the literature. For instance, in
rheumatoid arthritis, antigens that elicit autoantibodies include La,
Hsp65, Hsp70, type II collagen, hnRNP-B1, CCP, and Ro/La. Antigens
eliciting autoantibodies in multiple sclerosis include myelin
oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP),
protelipid protein (PLP), oligodendrocyte-specific protein (OSP), and
myelin-associated glycoprotein (MAG). Antigens in autoimmune thyroid
disease include thyroglobulin, TSH receptor, and thyroid peroxidase.
Thus, a peptide array can be made using any of the methods herein to
include a number of peptide clusters. The peptides on the array can
comprise the entire sequence of 50%, 90%, or all proteins encoded by
antigens that elicit an antibody response in subjects with an autoimmune
disease.

Peptide Arrays with Peptides for Research and Diagnostic Applications
Related to Viruses

[0125]In other embodiments, the peptide array contains peptides with
sequences from viral proteins. The viral proteins may be viral envelope
proteins from a viral family or from all viruses. The peptide sequences
may overlap. In addition, the peptides on the array may be antigenic
peptides covering multiple viral proteins, proteins from a viral family,
or proteins from all viruses. Viral proteins can include viral envelope
proteins and viral coat proteins, for example. Examples of virus families
include, for example, adenovirus, iridovirus, herpesvirus, papovavirus,
parvovirus, poxvirus, coronavirus, orthomyxovirus, paramyxovirus,
picornavirus, retrovirus, and rhabdovirus.

[0126]The peptides on the array can comprise the entire sequence of 50%,
90%, or all of the sequences of all viral envelope proteins of a viral
family or all viruses. The peptides on the array can comprise 50%, 90%,
or all of the sequences of overlapping antigenic peptides covering all
viral proteins of a viral family or all viruses.

[0127]The peptide arrays can also be made from peptide sequences from
viruses that can be used as bioterrorism agents, such as Variola major
virus, which causes small pox; encephalitis viruses, such as western
equine encephalitis virus, eastern equine encephalitis virus; and
Venezuelan equine encephalitis virus, arenaviruses, bunyaviruses,
filoviruses, and flaviviruses

Peptide Arrays with Peptides from Non-Viral Infectious Agents

[0128]Peptide arrays can also be made using peptide sequences from other
infectious agents or pathogens, including, for example, bacteria, fungi,
protozoa, multicellular parasites, and other microorganisms. The peptides
can be from prions. The peptide sequences can be from proteins from
bacteria that include, for example, Bacillus anthracis, Neisseria
meningitidis, Streptococcus pneumoniae, Staphylococcus aureus, Listeria
monocytogenes, Haemophilus influenzae, Mycobacterium tuberculosis,
Pseudomonas aeruginosa, Clostridium botulinum, Brucella abortus, or other
bacteria.

Peptide Arrays with Peptides with Random Sequences

[0129]Peptide arrays with random peptide sequences can be made. The
peptides with random sequence can be grouped into sub-libraries based on
the frequency with which they are present in a given proteome. For
instance, the 100, 200, 1000, or 10,000 most commonly occurring sequences
of 6-150 amino acids in the human proteome can be synthesized as peptides
on an array. The 100, 200, 1000, or 10,000 least commonly occurring
sequences of 6-150 amino acids in the human proteome can be synthesized
as peptides on an array.

Use of Peptide Arrays for Research Applications

[0130]Any of the peptides arrays described herein can be used as a
research tool. In one aspect of the invention, peptides arrays are used
for high throughput screening assays. For example, enzyme substrates
(i.e. peptides on a peptide array described herein) can be tested by
subjecting the peptide array to an enzyme and identifying the presence or
absence of enzyme substrate(s) on the array. Identifying the peptide can
be by detecting at least one change in said at least one peptide. More
than one change can also be identified.

[0131]The change detected can be any enzymatic reaction or process, for
example hydrolysis, proteolysis, dephosphorylation, phosphorylation or
complex formation between the enzyme and one or more of the substrates on
the array. Complex formation can also be used to determine the binding
specificity of the enzyme.

[0132]Enzymatic activity can be determined by various means. For example,
enzyme activity can be determined by applying the enzyme to a peptide
array described herein and determining a profile or signature of
enzymatic activity across a broad spectrum of substrates.

[0133]Enzymes screened or tested, or used for determining activity, can be
from cell lysates or purified proteins. Enzymes can be from prokaryotic
or eukaryotic cells. The enzymes can be purified proteins produced by
recombinant means or endogenous proteins. The enzymes can be any enzyme
known in the art, for example hydrolases or kinases.

[0134]Kinases can be screened using the peptide array. For example, as
shown in FIGS. 13A and B, enzymes such as a mixture of kinases, or a
single kinase, can be applied to a peptide array representing kinase
substrates. The substrates that are phosphorylated can then be detected.
Detection can be by fluorescence (see FIG. 14), for example, by using
commercially available reagents such as ProQ Diamond (Invitrogen, CA).
Binding assays can also be used with kinases and peptide arrays, wherein
either the kinase or the peptide is labeled, and binding affects the
level of fluorescence. Many tags are available for labeling, for example,
including, but not limited to, fluorescein, eosin, Alexa Fluor, Oregon
Green, Rhodamine Green, tetramethylrhodamine, Rhodamine Red, Texas Red,
coumarin and NBD fluorophores, QSY (Invitrogen), dabcyl and dabsyl
chromophores and biotin, as well as antigens or antibodies.
Phosphorylation can also be detected by mass spectrometry. Mass
spectrometry can include tandem mass spectrometry (MS/MS),
matrix-assisted laser desorption source with a time-of-flight mass
analyzer (MALDI-TOF), and liquid chromatography/mass spectrometry
(LC/MS). Phosphorylation can be detected using labeled ATP, such as
radiolabeled ATP. Antibodies specific for phosphorylation can also be
used for detection, or used to detect the bound kinase.

[0135]Identified peptides can serve as a tool to identify in vivo
substrates of the kinase or as possible drugs for the kinase. For
example, EC50 or substrate specificity can be determined by screening the
kinases with a peptide array (see for example, FIGS. 15, 16, and 17).
Substrate specificity can be determined for kinases within the same
family (for example, FIGS. 18, 19, and 20). Peptides identified can be
further tested as substrates for the kinase or inhibitors of the kinase.
Kinase inhibitors, such as candidate inhibitors, can also be screened
using the peptide arrays of the present invention, for example as shown
in used to determine the effect on kinase activity of different
inhibitors (see for example, FIGS. 21, 22, and 23).

[0136]In certain embodiments, hydrolases such as proteases, phosphatases,
lipases, and esterases are screened using peptide arrays of the present
invention. Cleaved peptides can be measured by having fragments detected
by mass spectrometry or by optical means such as fluorescence, wherein
the peptides on the array were labeled. For example, a protease can have
its activity measured by peptide cleavage, as shown in FIG. 24, wherein
the peptide is labeled with a fluorophore and cleavage measured by the
amount of fluorescence. Many tags are available for labeling peptides,
for example, including, but not limited to, fluorescein, eosin, Alexa
Fluor, Oregon Green, Rhodamine Green, tetramethylrhodamine, Rhodamine
Red, Texas Red, coumarin and NBD fluorophores, QSY (Invitrogen), dabcyl
and dabsyl chromophores and biotin. For example, as shown in FIGS. 25 and
26, the fluorescence intensity of the peptide array before and after
cleavage assays with trypsin (FIG. 26) and HIV-1 protease (FIG. 26).
Another assay for proteases or other proteins for substrate specificity
is through binding assays. The test protein can be labeled and binding
measured by determining the amount of label being bound and to which
peptide the test protein is binding, based on the location of the label.

[0137]Peptide arrays can also be used in simple screening assays for
ligand binding, to determine substrate specificity, or to determine the
identification of peptides that inhibit or activate proteins. For
example, peptides that bind signaling receptors involved in cell growth.
Labeling techniques, protease assays, as well as binding assays are well
known by one in the arts.

[0138]In yet another embodiment, phosphatases can be screened with the
peptide array. The peptide array used to screen phosphatases is one
having at least a subset if not all of its peptides be phosphatases
substrates. In one preferred embodiment, the subset or all of the
peptides synthesized on such array are selected from a publicly available
phosphobase such as http://www.cbs.dtu.dk/databses/PhosphoBase/ or
fragments thereof. Assays used may include binding assays and phosphatase
assays, both techniques being well known to one in the arts.

[0139]In another embodiment, antibodies are screened on the peptide array,
wherein the peptides of the array are epitopes. Epitopes for specific
antibodies are determined and can also be used to generate antibodies or
to develop vaccines.

[0140]In another example, the peptide array can be used to identify
biomarkers. Biomarkers may be used for the diagnosis, prognosis,
treatment, and management of diseases, including, but not limited to
diseases such as a disease associated with apoptosis, a disease
associated with signal transduction pathways of GPCRs, cancer, autoimmune
diseases, and infectious diseases. Biomarkers may be expressed, or
absent, or at a different level in an individual, depending on the
disease condition, stage of the disease, and response to disease
treatment. Biomarkers may be DNA, RNA, proteins (e.g., enzymes such as
kinases), sugars, salts, fats, lipids, or ions.

[0141]For example, an individual had a cancer biomarker which is an
antigen. The individual has a specific cancer, stage of cancer, or
response to certain cancer treatments. The individual's autoantibodies
are obtained through their serum and screened against variety of peptides
on a peptide array. The identification of specific peptides that bind to
autoantibodies also leads to the discovery of new biomarkers and provides
insight to the mechanism of the disease that causes generation of the
autoantibodies. In another embodiment, the binding of the autoantibodies
to specific peptides can create an "autoantibody signature". The
autoantibody signature is specific to a particular disease, stage of the
disease, or response to certain disease treatments. Thus, the
autoantibody signature can be useful in determining the diagnosis for
other individuals with a similar signature, or for example, including an
individual in a clinical trial.

[0142]The applications for research using peptide arrays is numerous and
information about enzyme/substrate, enzyme/inhibitor, antibody/antigen,
and protein/protein interactions can illuminate understanding of
biological processes leading to the drug discovery and development.

[0143]A peptide array can be used for epidemiology research. For example,
a peptide array can be used to determine the causative agent of a
disease. A sample from a patient with a disease can be applied to a
peptide array as described above, such as a peptide array containing
peptides with sequences from viruses, bacteria, or microorganisms.
Binding to the peptide array by antibodies produced by the patient to the
infectious agent can be used to determine identify the agent that caused
the disease.

[0144]The peptide array of the present invention can be used to study
antigen specific tolerance therapy and other immunoregulatory therapies.

Use of Peptide Arrays for Therapeutic Purposes

[0145]The methods of the present invention also provides for methods of
identifying bioactive agents. A method for identifying a bioactive agent
can comprise applying a plurality of test compounds to an ultra high
density peptide array and identifying at least one test compound as a
bioactive agent. The test compounds can be small molecules, aptamers,
oligonucleotides, chemicals, natural extracts, peptides, proteins,
fragment of antibodies, antibody like molecules or antibodies. The
bioactive agent can be a therapeutic agent or modifier of therapeutic
targets. Therapeutic targets can include phosphatases, proteases,
ligases, signal transduction molecules, transcription factors, protein
transporters, protein sorters, cell surface receptors, secreted factors,
and cytoskeleton proteins. For example, a therapeutic target can be a
kinase or GPCR. In other embodiments, the therapeutic target is a
molecule involved in DNA damage or apoptosis, such as those in FIG. 6 or
7. Therapeutic targets can include any molecule involved in a condition
or disease, for example, molecules involved in inflammation,
neurodegenerative diseases, or Alzheimer's disease, such as shown in FIG.
8 or 9.

[0146]In another aspect of the present invention, the peptide arrays are
used to identify drug candidates for therapeutic use. In one embodiment,
peptides identified by using peptide arrays in screening assays such as
those mentioned above for ligand binding to determine substrate
specificity can further be used to determine the peptide activity for a
given test substrate. For example, whether the peptide inhibits or
activates the activity of the test substrate. Peptides can screened as a
potential drug by determining if the peptides can inhibit an aberrant
active protein causing disease in an individual. An example is whether a
peptide identified as binding a kinase may inhibit kinase activity of the
given kinase. The peptide may then be used as a therapeutic agent, as
kinases are implicated in a number of conditions and disorders, such as
cancer. In another embodiment, wherein epitopes for specific antibodies
are determined by an assay mentioned above, the epitopes may be developed
as a drug to target antibodies in disease. Another embodiment is the
identification of ligands for receptors through the use of peptide
arrays, in which the peptides can then be used as a therapeutic against
diseases in which there is excessive receptor signaling causing diseases
such as cancer.

Use of Peptide Arrays for Medical Diagnostics

[0147]In one aspect, the present invention provides peptides arrays for
the use of medical diagnostics. The peptide array may be used in
determining response to administration of drugs or vaccines. For example,
an individual's response to a vaccine can be determined by detecting the
antibody level of the individual by using an array with peptides
representing epitopes recognized by the antibodies produced by the
induced immune response. Another diagnostic use is to test an individual
for the presence of biomarkers, samples are taken from a subject and the
sample tested for the presence of one or more biomarkers. For example, a
subject's serum can be used as a sample and the presence of a cancer
antigen, such as prostate-specific antigen (PSA) is used to diagnose
prostate cancer. However, in general, the current methods of using a
single biomarker for diagnosis of a condition is severely limited as many
biomarkers currently in use, such as PSA and carcinoembryonic antigen
(CEA), have limited sensitivity and specificity (Cho-Chung, Biochimica et
Biophysica Acta 1762 (2006) 587-591).

[0148]Multiple studies have shown that patient with cancer produce
detectable autoantibodies to certain tumor-associated antigens.
Autoantibodies are produced by individuals in an immune response to
cancer. Autoantibodies themselves can thus be used as biomarkers, and
detected by peptides specific to the autoantibodies. The peptide array
allows for better sensitivity and specificity in testing of biomarkers
and also allows for easy testing of a number of biomarkers with one
sample.

[0149]Biomarkers other than PSA and CEA, such as extracellular
cAMP-dependent protein kinase A (ECPKA), a normally intracellular protein
that is secreted in serum of cancer patients, can also be used.
Biomarkers that have been used that are not as specific or sensitive but
now may be useful in diagnosis with the use of peptide arrays include
serum oetopontin (previously implicated in lung cancer), p53 (used in the
diagnosis of pancreatic cancer), CEA (for the diagnosis of colon, lung,
breast, ovarian, bladder cancers), as well as tumor associated
glycoprotein-72 (TAG-72), carbohydrate antigen (CA19-9), lipid associated
sialic acid (LASA), alpha-fetoprotein (AFP, for the diagnosis of liver
cancer), CA125 (for the diagnosis of ovarian), CA15-3 (for the diagnosis
of breast cancer), human chorionic gonadotropin (hCG, for the diagnosis
of breast cancer), prostatic acid phosphatase (PAP, for the diagnosis of
a prostate cancer marker). (Cho-Chung, Biochimica et Biophysica Acta 1762
(2006) 587-591; Nesterova et al., Biochimica et Biophysica Acta 1762
(2006) 398-403). Other autoantibodies that may be detected by the present
invention include those in Table 1.

[0150]The biomarkers associated with the above cancers are not limited to
their use in the detection of that specific cancer. For example, a
plurality of autoantibodies can be recognized by a peptide array, forming
an autoantibody signature specific for prostate cancer. The
autoantibodies in the signature for prostate cancer diagnosis may include
autoantibodies that had previously been associated with biomarkers to
diagnose cancers not of the prostate. Autoantibodies to 22 peptides have
been identified in determining presence of prostate cancer and are better
at diagnosing prostate cancer in comparison to the conventional biomarker
of PSA (Wang et al. N. Engl. J. Med. (2005) 1224-1235). Peptides based on
the 22 sequences in Table 2, or a subset thereof, and are specifically
recognized by the autoantibodies that detect the sequences in Table 2,
are synthesized on an array. An individual's serum can then be used to
screen against the peptide array to determine a prostate cancer
diagnosis, prognosis, treatment, and management for the individual.
Prognosis may depend on the autoantibody signature and thus information
on the stage of the cancer may be determined, such as whether it affects
part of the prostrate, the whole prostate, or has spread to other places
in the body. Treatment and management of the cancer will vary depending
on the prognosis, examples being surgery, chemotherapy, hormone therapy,
cryosurgery, biologic therapy, radiation therapy, or high intensity
ultrasound therapy.

[0151]Autoantibodies produced by individuals in response to other
diseases, such as autoimmune diseases, inflammatory diseases,
cardiovascular diseases, metabolic diseases, and infectious diseases, can
be also detected by the peptide arrays of the present invention. For
example, peptides (e.g. epitopes) specific to the autoantibodies of
autoimmune diseases such as systemic lupus erythematosis (SLE),
scleroderma, rheumatoid arthritis (RA), or Sjogren syndrome, are produced
on an array. The resulting peptide array is then used in the detection of
an individual's autoantibodies, and thus, the diagnosis, prognosis,
treatment, and management of an individual's disease can be determined
based on the determination of an individual's autoantibodies. Similarly,
peptides specific to autoantibodies produced in infectious diseases are
used to determine the presence of an infectious agent in an individual,
stage of infection, etc.

[0152]A condition that can be diagnosed or prognosed with a peptide array
includes, for example, cancer, autoimmune disorder, an infectious
disease, an epidemic, transplant rejection, a metabolic disease, a
cardiovascular disease, a dermatological disease, a hematological
disease, a neurodegenerative disease, an inflammatory disease, and
infarctions (e.g. myocardial infarction, stroke).

[0163]FIG. 10 illustrates overlapping peptides that can be on an array for
investigating organ transplant rejection. An antibody epitope array can
be used to study organ transplant rejection. Up to 20 million organ
specific 9 mer peptides can be synthesized on an array, and samples from
subjects can be applied to the arrays to monitor organ specific global
antibody responses for diagnosis of rejection. An organ proteome can be
10,000 proteins, with each protein averaging approximately 350 amino
acids. Thus, approximately 350 9 mer peptides with one amino acid overlap
for each protein would total approximately 3.5×106 peptides
for one organ specific chip. Up to 20 million overlapping 9 monomer
peptides covering the full length of all known organ specific proteins
can be synthesized on an array. Examples of organs whose proteomes could
be used to design peptide arrays include the kidney, heart, liver. Other
embodiments of the array can contain all antigenic (antigenic peptides-B
cell epitopes) 9 mer peptides covering the full-length of all known organ
specific proteins. Another embodiment of the array contains all antigenic
(antigenic peptides-B cell epitopes) peptides covering the full length of
all proteins in the organ proteome. Proteins known to elicit antibodies
that are markers of transplant rejection include intermediate filament
vimentin, ribosomal protein L7, quadrature-transducin, 1-TRAF or
lysyl-tRNA synthetase (see U.S. Pat. No. 7,132,245). The presence of
human IgM antibodies that specific to a peptide or peptides on an organ
specific peptide array can indicate acute transplant rejection, and the
presence of human IgG antibodies specific to a peptide or peptides on an
organ specific peptide array can indicate chronic rejection.

Enzymatic Activity Profiling

[0164]The present invention further provides determining the enzymatic
activity of an enzyme using a peptide array described above. An enzyme
can be applied to the peptide arrays described herein, and the enzymatic
activity determined by detecting at least one change in at least one
peptide from the peptide array. For example, the activity of a kinase,
protease, phosphatase or other hydrolase can be determined. The activity
of a single enzyme, class of enzyme, or the entire enzyme family of an
organ or organism can be determined and an enzymatic activity profile
generated.

[0165]The peptides arrays can be used for generating profiles for an
organism. An enzymatic activity profile of an organism can be determined
by applying a biological sample from the organism to peptide array,
measuring the enzymatic level of the sample, and determining the
enzymatic activity profile for the organism. The organism can be
prokaryotic, for example such as bacteria. The organism can be eukaryotic
such as yeast. Other eukaryotes can include humans and non-humans, such
as animal models including mice, rats, birds, cats, dogs, sheep, goats,
and cows. Biological samples can cell lysates or tissue samples. Samples
can be obtained from the organism by a number of methods known in the
arts.

[0166]Enzymatic profiles can be generate for a single type of enzyme, a
number of enzymes, or an entire class of enzymes, or all enzymes from a
biological sample. Enzymatic profiles can be generated for any enzyme,
such as hydrolases or kinases. For example, an enzymatic profile can be
for a single kinase, such as protein kinase C. In other embodiments, an
enzymatic profile can be generated for a family of kinases, such as all
cyclin-dependent kinases. In yet another embodiment, an enzymatic profile
can be generated for a kinome, generating a kinome activity profile. A
kinome activity profile can be generated by applying a biological sample
from an organism to an ultra high density peptide array and measuring the
level of phosphorylation of the peptide array.

[0167]The enzymatic profiles can be used for a multitude of purposes, such
as diagnosing any of the diseases mentioned herein. For example, a
biological sample from a subject can be applied to a peptide array,
wherein the peptide array comprises a plurality of peptides coupled to a
support, and a set of said peptides are hydrolase or kinase substrates,
detecting the enzymatic activity of said sample on said peptide array;
and, diagnosing a disease state in the subject.

[0168]The enzymatic profiles can also be used for determining the toxicity
or efficacy profile of a subject. For example, a kinome activity profile
can be used to determine the toxicity or efficacy profile of a subject.
For example, a toxicity profile or an efficacy profile of a drug may be
generated for a subject prior to administration of a drug or being on a
particular regimen. A toxicity or efficacy profile of one or more drugs
can be determined for a subject by applying a biological sample from a
subject to an ultra high density peptide array. The toxicity or efficacy
profile can be compared to control profiles, such as profiles from
controls subjects that have responded well to the drug, or control
subjects who have responded negatively to the drug, to determine how the
subject may respond to the drug. The toxicity or efficacy profiles can be
used to determine whether alternative drug treatments may provide better
efficacy and fewer side effects or toxic effects at higher dosages.
Toxicity or efficacy profiles can also be generated after a subject has
been administered the drug. The profiles can be used in pre-clinical
studies, for example with animal models, or be used in clinical studies,
for example with humans.

[0169]The profiles can also be used to monitor the efficacy or toxicity of
a drug in a subject. A first biological sample from a subject prior to
administration of a drug can be applied to a first ultra high density
peptide array, and a second biological sample from the subject after
administration of a drug to a second ultra high density peptide array is
applied. The first and said second peptide arrays can be used to generate
enzymatic activity profiles and compared to monitor the toxicity or
efficacy of said drug. Various treatment regimens, such as varying
dosage, number of dosages, time between dosages, and different
administration routes can be tested and profiles generated based on the
various treatment regimens to determine the toxicity or efficacy of a
drug.

[0170]The enzymatic activity profiles, such as the kinome activity
profile, can also be used to stratifying a subject within a patient
group. A biological sample from a subject can be applied to peptide
array, the enzymatic activity profile for the subject is compared to
enzymatic profiles of different subject groups, and based on the
comparison, the subject is stratified into a treatment group. The
enzymatic activity profiles can also be used for diagnosing or prognosing
a subject, for example with a condition or disease such as cancer,
inflammatory disease, neurodegenerative disease, or Alzheimer's.

Use of Peptide Arrays to Stratify Patients into Treatment Groups

[0171]Peptide arrays can also be used to stratify patient populations
based upon the presence of a biomarker that indicates the likelihood a
subject will respond to a therapeutic treatment. One example of patient
stratification relates to the use of Herceptin® in treating breast
cancer patients. Breast cancer patients respond differently to treatment
with Herceptin® based on their HER-2 levels. Breast cancer patients
with overexpression of HER-2 respond to treatment with Herceptin®,
whereas patients that do not overexpress HER-2 do not respond to
Herceptin® treatment. Thus, HER-2 is a critical biomarker for
stratification of breast cancer patients into treatment groups for
Herceptin®. The peptide arrays of the present invention can be used
to identify known biomarkers to determine the appropriate treatment
group. For instance, a sample from a subject with a condition can be
applied to an array. Binding to the array may indicate the presence of a
biomarker for a condition. Previous studies may indicate that the
biomarker is associated with a positive outcome following a treatment,
whereas absence of the biomarker is associated with a negative or neutral
outcome following a treatment. Because the patient has the biomarker, a
health care professional may stratify the patient into a group that
receives the treatment.

[0172]In one aspect, the present invention contemplates a method for
selecting therapy for a subject comprising: applying a sample from said
subject to a peptide array; determining the enzymatic activity of said
sample by detecting at least one change in at least one peptide from said
peptide array, and selecting a therapy for said subject from determined
enzymatic activity. The selecting a therapy step can comprise selecting a
drug treatment, wherein the drug is a kinase inhibitor drug, a GPCR drug,
an apoptosis targeting drug, neurodegenerative inhibiting drugs, or a
drug targeting DNA damage repair. The subject may have a condition
associated with abnormal activation of the apoptosis pathway, DNA damage
repair pathway, signal transduction pathways of GPCRs, or a
neurodegeneration. Examples of kinase inhibitor drugs contemplated herein
include Gleevac, Dasatinib and SKI606. Examples of GPCR drugs include
Zyprexa®, Clarinex®, Zantac®, and Zelnorm®. Examples of
neurodegenerative inhibiting drugs include
(-)-epigallocatechin-3-gallate, penserine, R-BPAP, flurbiprofen, or an
AChE inhibitors. Examples of apoptosis targeting drugs are bortezomib,
CCI-779, and RAD 001. An example of a DNA repair pathway drug is
Trifluoperazine. In some instances, selecting a therapy step further
comprises determining a treatment regimen for said subject. In some
instances, selecting a therapy step comprises determining a dosage level.
A peptide array used for therapy selection can be any of the ones
described herein, including those having at least 5,000 different
peptides.

Use of Peptide Arrays for Biodefense

[0173]A peptide array of the present invention can also be used for
biodefense. Biodefense can involve generating vaccines against diseases
that can be caused by bioterrorism agents, developing diagnostic tests to
rapidly identify exposures to bioterrorism agents and allow for the
determination of appropriate treatments, and providing therapies to
patients that have been subjected to a bioterrorism attack.

Business Methods Relating to Peptide Arrays

[0174]The present invention contemplates business methods that produce and
manufacture peptide arrays having the features described herein. For
example, in some cases a peptide array is one produced using
photolithography using photoresist and RAC or other means described
herein. In some embodiments, the peptide array is produced or
manufactured without a mask. In some embodiments, the peptide array is
produced using an electrochemical reagent and methods.

[0175]The methods of the present invention includes manufacturing a
peptide array comprising applying photoresist to a plurality of monomers
on a support; removing the photoresist in selected regions using
photolithography, for example, with the use of a mask or micromirrors;
causing acid or base labile protecting groups to be removed form the
monomers indirectly; delivering monomers to the array to generate a
plurality of peptides whose sequences have a hydrolase site or
phosphorylation site at a different position than the other sequences in
the peptide cluster. In some embodiments, the peptide sequences overlap
to form a common protein sequence with at least one enzymatic reaction
site, such as a protease, phosphatase, or phosphorylation site. The
peptides can be substrates for at least 50% of the proteases of an organ
or an organism, at least 50% of the phosphatases of an organ or an
organism, at least 50% of the kinases of an organ or an organism, or the
entire kinome of an organ or organism. The peptides can be substrates for
a pathway, such as proteins downstream of a G-protein coupled receptor.

[0176]Such peptide array can have any of the features described herein.
For example, each region or feature can be between 0.2 to 100 um2,
0.2 to 10 um2, 0.2 to 1 um2, 0.2 to 0.5 um2, or up to 0.5,
1, 5, 10, 15, 20, 25, 50, 100, 250, 500, 1000 um2. The array can at
least 20, 100, 200, 300, 400, 800, 1000, 1500, 2000, 3000, 4000, 5000,
10,000, 20,000, 50,000, 75,000, 100,000, 150,000, 200,000, 300,000,
400,000, 500,000, 1,000,000, 2,000,000, 2,250,000, 5,000,000, 10,000,000,
or 100,000,000 unique peptides on a single array. The peptide arrays can
also have substrate peptide clusters.

[0177]The business method above can provide the above arrays for consumers
for research and diagnostic purposes. A business method herein provides a
service in exchange for a fee to customers whereby a sample is sent to
the business for research or diagnostic purposes, and the business
analyzes the sample using one or more of the peptide arrays described
herein and sends a report to the customer with analysis of the sample.
The business than provides information about the sample to the customer.
The information can be a diagnostic, e.g., whether a patient has a
condition such as cancer, Alzheimer's, an autoimmune disorder, etc. The
information can be provided to a customer to stratify or select patients
for a clinical study, e.g., whether the patient is susceptible to drug
toxicity. The information can also provide a general health monitoring
tool to a doctor by providing an enzyme profile or kinase profile (finger
print) or research information.

[0178]While preferred embodiments of the present invention have been shown
and described herein, it will be obvious to those skilled in the art that
such embodiments are provided by way of example only. Numerous
variations, changes, and substitutions will now occur to those skilled in
the art without departing from the invention. It should be understood
that various alternatives to the embodiments of the invention described
herein may be employed in practicing the invention. It is intended that
the following claims define the scope of the invention and that methods
and structures within the scope of these claims and their equivalents be
covered thereby.

Characteristics of the Features of the Peptide Arrays

[0179]An array of the present invention can comprise hundreds, thousands,
or millions of features. A feature is a localized area on a support which
is, was, or is intended to be used for formation of a selected polymer or
polymers. A feature may have any convenient shape, e.g., circular,
elliptical, wedge-shaped, linear, or rectangular, such as a square.
Feature sizes can be up to approximately 0.5, 1, 2.5, 5, 10, 15, 20, 25,
50, 100, 250, 500, 1000, or 10,000 um2 or between 0.2 to 100 um2,
0.2 to 10 um2, 0.2 to 1 um2, or 0.2 to 0.5 um2. Smaller
features allow for increased numbers of features per given support size.
For example, a peptide array manufactured by the methods herein can have
at least 20, 100, 200, 300, 400, 800, 1000, 1500, 2000, 3000, 4000, 5000,
10,000, 20,000, 50,000, 75,000, 100,000, 150,000, 200,000, 300,000,
400,000, 500,000, 1,000,000, 2,000,000, 2,250,000, 5,000,000, 10,000,000,
or 100,000,000 features on a single support. For example, the numbers of
features on a 6×6 mm2 array can be at least 14,400, 57,600,
90,000, 160,000, or 360,000. The number of features on a 1.5×1.5
cm2 array can be at least 225, 900, 3,600, 22,500, 90,000, 360,000,
562,500, 1,000,000, 2,250,000, 10,000,000, or 100,000,000.

[0180]The number of copies of a peptide within a feature can be from at
least 1 to at least 10. In some embodiments, at least 100 peptides are
located within a feature. In some embodiments, the number of peptides in
a feature can be in the thousands to the millions. Within features, the
peptides synthesized therein are preferably synthesized in a
substantially pure form. In some instances, only up to 50%, 60%, 70%, or
80% of peptides within a feature are identical to a predetermined
sequence.

[0181]At least a subset of features comprises peptides with sequences as
in another feature on the same array. In the alternative, at least a
subset of features in an array can comprise peptides whose sequences are
different than the peptide sequences of the other features. A single
peptide array can also have features that have the same peptide sequence
as other features, as well as features with a different peptide sequence
than other features. In some embodiments, each of the features on a
peptide array can comprise a different sequence. For example, a peptide
array manufactured by the methods herein can have at least 20, 100, 200,
300, 400, 800, 1000, 1500, 2000, 3000, 4000, 5000, 10,000, 20,000,
50,000, 75,000, 100,000, 150,000, 200,000, 300,000, 400,000, 500,000,
1,000,000, 2,000,000, 2,250,000, 5,000,000, 10,000,000, or 100,000,000
different peptide sequences on a single array. The feature density on an
array can be greater than 100,000, 500,000, 1,000,000, 50,000,000, or
100,000,000/cm2. The array can have dimensions of such as those of
any known nucleic acid array, including 6×6 mm2 or
1.5×1.5 cm2.

[0182]In some instances at least 1%, 5%, 10%, 25%, 50%, 75%, 85%, 90%, or
99% of the peptides on the array may have a different sequence, i.e.,
sequence different from all other sequences on that same array. For
example, a peptide array made using photolithography can have peptides
with more than 100,000, 150,000, 200,000, 500,000, 1,000,000, 2,000,000,
10,000,000, 20,000,000, or 100,000,000 different sequences.

[0183]At least a subset of peptide(s) on an array can have a different
number of monomers from the other peptides. In the alternative, at least
a subset of peptides on an array can have the same number of monomers.
For example, a peptide array can have at least a subset of peptides or
all peptides with between 2 to 150 monomers, 3-50 monomers, or 4-10
monomers. At least a subset of peptides or all peptides can have 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomers.

[0184]An array with 400 peptides is generated using photoresist-RAC
technology wherein each peptide is approximately 9 amino acids long. The
peptides are designed to mimic epitopes to antibodies or mutants of the
corresponding epitopes, the mutants being unable to bind the antibodies.
Binding assays, detection sensitivity, CV, and linear dynamic range are
determined with the peptide arrays using standard techniques known in the
art. Results are compared to ELISA and are equivalent in sensitivity and
accuracy.

Example 2

Detection of Autoantibodies in Prostate Cancer

[0185]Peptides based on the sequences of Table 2 are synthesized on an
array using photoresist-RAC technology. Serum from a control group and a
group with prostate cancer are taken and screened with the peptide array.
Percentage of peptides bound is determined between the control group and
cancer group. Results are compared to results from peptide phage display
as described in Wang et al. N. Engl. J. Med. (2005) 1224-1235 and
determined to be equivalent.

Example 3

Peptide Array and Kinase Assay for Abl and Src Kinases

[0186]Peptide sequences as depicted in FIG. 16 were produced on a support
in the pattern shown, using methods as described in Examples 1 and 2. The
wild-type (WT) peptides substrates are recognized by their respective
kinase. A mixture of Src and Abl kinase was applied to the peptide arrays
comprising sequences 1-6. The EC50 for Src was shown to be ˜1.5
ng/μl (FIG. 15), the dynamic range approximately 0.1˜10
ng/μl, and a mixture of Src with Abl kinase did not interfere with the
kinase activity of either of the individual kinases, as shown in FIG.
13B.

[0188]PKA kinase (kinase reaction buffer as shown in Table 5, variations
of the buffers in Tables 11-13 are used depending on the specific kinase)
and PKB kinase belong to the same kinase family. The individual kinases
were applied to peptides arrays comprising the same peptide sequences in
the same configuration.

[0189]PKA and PKB have different activity against specific peptide
substrates as differences in the peptide detection was determined (for
example, the squared boxes highlighted in FIG. 18). The kinases show a
difference in preferred specificity in position -4 (4 amino acids shifted
from the phosphorylation site, Serine "S"), -1 (one position from
phosphorylation site), and +1 (one position from the serine).

[0190]PKC was applied to another peptide array with the same peptide
sequences in the same configuration as those used for PKA and PKB. PKC
has a different sequence preference in comparison to PKA and PKB (FIG.
19). PKC shows a different preference in position -4 (4 amino acids
shifted from the phosphorylation site, Serine "S") and +1 (one position
from the serine).

[0191]The positional preference of the AGC family kinases PKA, PKB, and
PKC are shown in FIG. 20. The preference was based on relative signal
intensity over kemptide (or peptide). The bolded residues are from
previously published work whereas the other residues were not published.

[0192]The ATP competitive inhibitor, staurosporin ("Stau.") was used in an
Src kinase inhibitor assay. A peptide array with Src kinase substrates
was produced. A kinase assay was performed using Src with Staurosporin.
Staurosporin inhibited Src kinase activity by up to 80%. The IC50 was
estimated to be approximately 450 nM in the presence of 2 uM ATP (FIG.
21). The IC50 of Staurosporin on Src kinase is comparable to the 200-400
nM reported in the literature.

Example 6

Gleevac Inhibition of Abl Kinase

[0193]Gleevac, a commercially available kinase inhibitor, has specific
bioactivity on various forms of Abl kinase. Gleevac inhibits active Abl
kinase. Gleevac was used in a kinase inhibitor assay with Abl and Src
kinase (FIG. 22). Gleevac inhibition of phosphorylated Abl kinase, non
phosphorylated Abl kinase, and Src kinase, or both, was tested using
peptide arrays with Abl and Src substrates as in Example 3. Kinase assays
and peptide arrays were as described in Example 6, but with the addition
of Gleevac in the kinase assay, and either phosphorylated or
non-phosphorylated Abl kinase and Src.

[0194]Leevac does not have an effect on phosphorylated Abl kinase nor Src
kinase activity (FIG. 22A). The percent inhibition of Gleevac, ˜75%
Gleevac inhibition (see FIG. 22E) is consistent with other commercial
assays

Example 7

Different Kinase Inhibitors in Kinase Inhibition Assay

[0195]The peptide array with Abl and Src peptide substrates as described
in Example 3 was used with various kinase inhibitors. Gleevac, Dasatinib
and SKI606 were used in kinase assays with Abl and Src kinase. Gleevac is
an active Abl kinase inhibitor, Dasatinib is a dual specific inhibitor,
and SKI-606 is an Src kinase inhibitor. As shown in FIG. 23, the peptide
arrays subjected to kinase assays with Src and Abl kinases and one of the
three inhibitors demonstrated the expected specificity of the kinase
inhibitor for their respective kinase.

Example 8

Kinase Substrate Array

[0196]Peptide sequences that are phosphorylated are obtained from the
phosphobase http://www.cbs.dtu.dk/databases/PhosphoBase/. Mutation
sequences are determined by single site scan through 20 natural amino
acids. The sequences obtained from the phosphobase covers 160 kinases, 52
tyrosine kinases, and 108 serine/threonine kinases. Approximately 1184
peptide sequences are synthesized on the array using photoresist-RAC
technology and each peptide comprises approximately 9 monomers. The
peptides represent 629 proteins covering ˜500 human intracellular
and surface kinases.

Example 9

Varying Phosphorylation Site Peptide Clusters

[0197]A subset of peptides on a peptide array is synthesized on a peptide
array using photoresist-RAC technology. The subset of peptides is in a
substrate peptide cluster. Each peptide in the peptide cluster is
approximately 9 monomers long and each peptide in the cluster has a
single Ser. The single Ser is in position 1 of one peptide, and shifted
one monomer position in the subsequent peptides within the cluster such
that each peptide in the cluster has a unique sequence (FIG. 7). Each
peptide has a unique sequence but the same amino acids, for example, each
peptide in the cluster can have 1 Ser, 2 Ala, 3 Gly, 1 Glu, 1 Phe, and 1
Asp, and the amino acid sequence is the same between peptides except for
the Ser and the amino acid in the position it is occupying in the
specific peptide. For example, peptide 1 has Ser in position 1 (P1-Ser),
and P2-Ala, P3-Ala, P4-Gly, P5-Gly, P6-Gly, P7-Glu, P8-Phe, and P9-Asp.
Peptide 2 has P1-Ala (P2 amino acid of peptide 1), P2-Ser, and P3 to P9
is the same as peptide 1 in the cluster. Peptide 3 will have P1-Ala (P3
amino acid of peptide 1), P3-Ser, and the remaining P2, P4-P9 are the
same amino acids in the same position as in peptide 1. The remaining
peptides in the cluster, peptides 4-9 will have Ser in the P4, P5, P6,
P7, P8, and P9, respectively.

Example 10

Constant Phosphorylation Site Peptide Clusters

[0198]A subset of peptides on a peptide array is synthesized on a peptide
array using photoresist-RAC technology. The subset of peptides is in a
substrate peptide cluster. Each peptide in the peptide cluster is
approximately 9 monomers long and each peptide in the cluster has a
single Thr. The Thr is in the same monomer position as all the other
peptides in the peptide cluster (FIG. 8). The remaining monomer positions
are filled with one of the remaining 17 amino acids. The number of
peptides is 136 peptides to encompass all the different variations.

Example 11

Kinome Activity Profile

[0199]A peptide array with substrates of the human kinome is produced
using photoresist technology. The peptide array has at least one
substrate for each kinase in the human kinome. A tissue sample from a
subject is taken and applied to the peptide array. The level of
phosphorylation from the tissue sample is determined and a kinome
activity profile generated for the subject. The kinome activity profile
can be used for diagnosis or prognosis of a condition, such as cancer.

Example 12

Peptide Cleavage Assay with Trypsin

[0200]A peptide array with the peptide sequence depicted in FIG. 25, a
substrate for trypsin, was produced by methods as described in Examples 1
and 2. The bolded portion is the trypsin cleavage site. The peptide was
fluorescently labeled with TAMRA (5-carboxytetramethylrhodamine,
available from Invitrogen) and coupled to a silicon support. The amount
of fluorescence before and after treatment of the peptide array with
trypsin was determined (FIG. 25). After cleavage, the amount of
fluorescence decreased as expected.

Example 13

Peptide Cleavage Assay with HIV-1 Protease

[0201]A peptide array with the peptide sequence depicted in FIG. 26, a
substrate for HIV-1 protease, was produced by methods as described in
Examples 1 and 2. The bolded portion is the HIV-1 protease cleavage site.
The peptide was fluorescently labeled with TAMRA
(5-carboxytetramethylrhodamine, available from Invitrogen) and coupled to
a silicon support. The amount of fluorescence before and after treatment
of the peptide array with HIV-1 protease was determined (FIG. 26). After
cleavage, the amount of fluorescence decreased as expected.

Example 14

(Prophetic) Diagnosis of Alzheimer's disease

[0202]A peptide array with peptides covering the proteome of a human is
used. Serum samples from subjects with Alzheimer's disease and subjects
without Alzheimer's disease are applied to peptide arrays of the same
configuration. A binding pattern (autoantibody signature) or a single
biomarker is searched for that is characteristic of subjects with
Alzheimer's disease and not subjects without Alzheimer's disease. A
sample from a subject with a condition is applied to a peptide array of
the same configuration. The binding pattern or the sample of the subject
is compared to the binding pattern of subjects with Alzheimer's and
subjects without Alzheimer's to determine if the subject has Alzheimer's
disease.

Example 15

(Prophetic) Human Antibody Epitope Array

[0203]The human genome has approximately 30,000 genes. The average length
of a protein encoded by a gene is 350 amino acids. Thus, 342 peptides of
nine amino acids are needed per protein to have an eight amino acid
overlap. Thus, 342×30,000=10,260,000 peptides are synthesized on a
support to cover the whole human proteome. For a 3 amino acid overlap,
(342/6)=1,7100,000 peptides are synthesized on a support.

Example 16

Peptide Synthesis on Glass or Silicon Surface

Preparation and Silanation

[0204]A solid support, plain glass (dimension: 1×3 inches,
thickness: 0.9-1.1 mm, Corning 2947) or silicon (dimension: 1×3
inches, thickness: 725 μm, SVM) slide or surface, was cleaned by
dipping in piranha solution (100 ml of 30% H2O2 with 100 mL of
H2SO4) for over 30 minutes with shaking. The slide was then
washed with deionized water, 3 times for 5 min each (shaking each time).
The slide was then washed with 95% ethanol, once for 5 min with shaking.
The oven is turned on and set to 110° C. The slide are transferred
into 0.5% APTES solution (1 mL of 3-aminopropyl-triethoxysilane (APTES)
with 199 mL of 95% ethanol) and for 30 min with shaking. The slide was
then washed with 95% ethanol, once for 5 min (with shaking), then washed
with isopropanol once for 5 min shaking. The wafer was then dried with
N2 in the oven at 50° C.

[0205]The slide was then transferred and cured at 100-110° C. in
N2 atmosphere oven for 60 min. The slide was then placed into a
vacuum chamber filled with N2. This was repeated twice.

Glycine Coupling

[0206]Next the slide was derivitized with glycine. The surface was
neutralized with 5% (v/v) diisopropyl ethyl amine
(DIEA)/dimethylformamide (DMF) for 5 min by dipping the slide in a DIEA
bath. The slide was then washed with DMF twice for 5 min each and then
with 1-methyl-2-pyrrolidone (NMP) twice for 5 min each.

[0207]The slide was then transferred to AA coupling solution (Table 6a and
6b) bath for 1 hour with shaking at room temperature.

[0208]The solution was replaced with 2% acetic anhydride/DMF for 30 min
with shaking at room temperature. The slide was then washed with DMF
twice, 5 min each, with isopropanol (IPA) twice, 5 min each. The slide
was then rinsed with IPA, dried with N2 in the oven at 50° C.
and then stored in a petri dish at room temperature.

Fluorescein Staining

[0209]Boc was removed by treating the slide with trifluoroacetic acid
(TFA) for 15 min, then washed with IPA 3 times, then washed with DMF for
5 min. The slide was then dipped into 5% (v/v) DIEA/DMF for 5 min, washed
twice with DMF, twice with NMP and rinsed with IPA. A polymethacrylate
(PMA) gel with pierced circles was placed on one side of the slide.
Thirty microlitres of FI-AA coupling solution (Table 7) was added in each
well and then covered with aluminum foil to protect from light for two
hours.

[0210]The FI-AA coupling solution from wells was removed and the wells
washed twice with NMP. The PMA gel was removed and the slide rinsed with
NMP, IPA and ethanol. The slide was dipped into 50% EDA/EtOH for 30 min,
washed twice with EtOH, 15 min each time. The slide was then rinsed with
IPA and dry with N2. Next, 1 drop of TE buffer, pH 8 was added and
covered with a cover slip. The slide was scanned for fluorescence on a
confocal microscope at 494 nm/525 nm (Ex/Em) and 0.4 gain. Images were
processed with Scion software. Background substracted intensity should be
˜100.

Synthesis Cycle

[0211]1. Boc deprotection & wash: Boc was removed by TFA or by PGA. For
TFA Boc removal, the slide was treated with 100% TFA for 30 min and then
washed with IPA 4 times and DMF once. For PGA Boc removal, the slide was
placed on the spinner and washed with acetone and isopropanol, program 2
(2000 rpm, 30 sec). PAG solution (1 ml, Table 8) was added and spin
coated as described in Example 2.

[0216]The deprotection solution was removed and the slide washed with IPA
4 times and then dried with N2.

[0217]Prior to a bio-assay, the slide is neutralized with 5% DIEA as
described above.

Example 17

Spin Coating and Exposure of Array

[0218]An array was spin coated and exposed by using a mask (EV620) or with
micromirrors. Spin coating was performed as in Table 10. Exposure with a
mask was performed as in Table 11, or with micromirrors as described in
Table 12.

TABLE-US-00012
TABLE 11
Exposure with Masks
Step Description Procedure
1 Intensity check Remove wafer chuck and replace with intensity
measurement
plate
Make sure OAI meter is adjusted to 365 nm wavelength
Place glass plate (with or without transparency) on top of the
circular mask opening
Place intensity probe on center of plate
Select "Uniformity Measurement" under the pop-down menu,
and follow step-by-step instruction on screen
After taking note of intensity reading, click "continue" on the
screen, then Exit
Remove glass plate
Remove measurement plate and replace with wafer chuck
2 Mask loading Place mask loading plate on top of wafer chuck
Place mask (or glass plate with transparency) on the mask plate
(press against the positioning pins)
Open the recipe file and enter the exposure time (Time =
Dose/Intensity)
Click on "Run" and follow step-by-step instruction on the
screen.
After mask is loaded, remove the mask loading plate from the
wafer chuck
3 Exposure Follow instruction on screen after mask is loaded to expose
substrate
Click "Exit" to remove mask and exit the program

TABLE-US-00013
TABLE 12
Exposure with Micromirrors
Step Description Procedure
1 Intensity check Make sure OAI meter is adjusted to 365 nm wavelength
Load the Bitmap file "Exposure Intensity Check" on the
computer screen
Place the intensity probe underneath the exposure field (use
sensor #1)
Press the Expose button and note the intensity reading
2 Substrate alignment Set the desired exposure time (Time =
Dose/Intensity)
and exposure Place substrate on vacuum chuck (press against the top left
hand corner) and turn on vacuum switch
Run the Labview program
Using the stage controller and the rotational stage knob, align
the crosshairs (or left corner and right edge) on the substrate to
line up with the crosshairs on the Labview display
Reset the X and Y coordinates on the remote display to zero
(press "UP" until "Clear All Axis Position" is displayed and
press "PGM"
On the computer display, switch from Labview program to
desired Bitmap artwork
Using the stage controller, move the stage to the correct X and
Y locations, then press the Expose button, repeat as necessary

[0219]While preferred embodiments of the present invention have been shown
and described herein, it will be obvious to those skilled in the art that
such embodiments are provided by way of example only. Numerous
variations, changes, and substitutions will now occur to those skilled in
the art without departing from the invention. It should be understood
that various alternatives to the embodiments of the invention described
herein may be employed in practicing the invention. It is intended that
the following claims define the scope of the invention and that methods
and structures within the scope of these claims and their equivalents be
covered thereby.